Preventing cytokine release syndrome

ABSTRACT

This document relates to methods and materials for preventing cytokine release syndrome (CRS). For example, methods and materials for using one or more catecholamine inhibitors to prevent a mammal from developing CRS are provided.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Patent Application Ser. No.62/610,620, filed on Dec. 27, 2017. The disclosure of the priorapplication is considered part of (and is incorporated by reference in)the disclosure of this application.

STATEMENT REGARDING FEDERAL FUNDING

This invention was made with U.S. government support under grant No.CA062924 from the National Institutes of Health. The U.S. government hascertain rights in the invention.

BACKGROUND 1. Technical Field

This document relates to methods and materials for treating and/orpreventing cytokine release syndrome (CRS). For example, this documentprovides methods and materials for using one or more catecholamineinhibitors to prevent a mammal from developing CRS.

2. Background Information

Inflammation is crucial for the defense against pathogens. However, whenuncontrolled, the cytokines that normally mediate protective immunityand promote recovery can themselves cause a dangerous systemichyperinflammatory state, also referred to as cytokine release syndrome(CRS) or cytokine storm, which can lead to cardiovascular collapse,multiple organ dysfunction and ultimately death (Kopf et al., 2010 Nat.Rev. Drug Disc., 9:703-18; Medzhitov, 2008 Nature, 454:428-35; Nathan,2002 Nature, 420:846-52; Rittirsch et al., 2008 Nat. Rev. Immunol.,8:776-87; van der Poll et al., 2017 Nat. Rev. Immunol., 17:407-20; andWiersinga et al., 2014 Virulence, 5:36-44). In addition to infections bynaturally occurring pathogens as in sepsis, CRS is also observed aftercertain biologics and/or immunotherapeutics are administered toexperimental animals or patients. These include oncolytic viruses andbacteria (Rommelfanger et al., 2013 Mol. Ther., 21:348-57; and Agrawalet al., 2004 PNAS USA, 101:15172-7), antibodies to cells or solublecomponents of the immune system (Suntharalingam et al., 2006 New Eng. J.Med., 355:1018-28; Ferran et al., 1990 Eur. J Immunol., 20:509-15; andHansel et al., 2010 Nat. Rev. Drug Disc., 9:325-38), cytokines (Panelliet al., 2004 J Transl Med, 2:17-31), and T-cells designed to kill cancercells (Teachey et al., 2016 Can. Disc., 6:664-79; Fitzgerald et al.,2017 Crit. Care Med., 45:e124-e31; Grupp et al., 2013 New Eng. J Med.,368:1509-18; Lee et al., 2014 Blood, 124:188-95; and Maude et al., 2014New Eng. J. Med., 371:1507-17). In fact, the major dose-limitingtoxicities of modern biotherapeutic agents can be attributed to theexcessive cytokine release, thereby seriously limiting the utility ofthese otherwise promising agents.

SUMMARY

This document provides methods and materials for treating and/orpreventing CRS. For example, this document provides methods andmaterials for administering one or more catecholamine inhibitors toprevent a mammal from developing CRS. For example, this documentprovides methods and materials for administering one or morecatecholamine inhibitors to prevent CRS in a mammal at risk ofdeveloping CRS.

As demonstrated herein, catecholamines orchestrate an immunedysregulation via a self-amplifying loop in immune system cells, andcatecholamine inhibitors (e.g., ANP, metyrosine, and/or prazosin) can beused to suppress catecholamine synthesis. Pharmacologic inhibition ofcatecholamine synthesis protected mice from the lethal complications ofCRS resulting from infections and various biotherapeutic agentsincluding oncolytic bacteria, antibodies, and CAR-T cells. Having theability to prevent CRS by disrupting a catecholamine synthesis loopprovides a unique and unrealized opportunity to treat and/or preventlife-threatening toxicities associated with therapies withbiotherapeutic agents.

In general, one aspect of this document features a method for preventingcytokine release. The method includes, or consists essentially of,administering a catecholamine inhibitor to a mammal identified as beingat risk of developing CRS. The CRS can be associated with sepsis. TheCRS can be associated with an immunotherapy (e.g., orthoclone OKT3,muromonab-CD3, rituximab, alemtuzumab, tosituzumab, CP-870,893,LO-CD2a/BTI-322, TGN1412, tisagenlecleucel, axicabtagene ciloleucel,bi-specific T-cell engagers (BiTEs), adoptive T-cell therapy, dendriticcell therapy, interferon therapy, interleukin therapy, bacterialtherapy, and/or viral therapy). The immunotherapy can be a cancerimmunotherapy. The immunotherapy can be for treating an autoimmunedisease (e.g., rheumatoid arthritis, juvenile idiopathic arthritis,ankylosing spondylitis, psoriasis, systemic lupus erythematosus, celiacdisease, type 1 diabetes, autoimmune encephalomyelitis, multiplesclerosis, central nervous system autoimmune demyelinating diseases,chronic inflammatory demyelinating polyneuropathy, transverse myelitis,polymyositis, dermatomyositis, Crohn's disease, ulcerative colitis,autoimmune hemolytic anemia, autoimmune cardiomyopathy, autoimmunethyroiditis, Graves' disease, Sjogren's syndrome, Goodpasture syndrome,autoimmune pancreatitis, Addison's disease, alopecia, myasthenia gravis,sarcoidosis, scleroderma, pemphigus vulgaris, mixed connective tissuedisease, bullous pemphigoid, or vitiligo). The mammal can be a human.The catecholamine inhibitor can include a tyrosine hydroxylase inhibitor(e.g., metyrosine). The catecholamine inhibitor can include anatriuretic peptide (e.g., atrial natriuretic peptide (ANP), brainnatriuretic peptide (BNP), C-type natriuretic peptide (CNP), anddendroaspis natriuretic peptide (DNP)). When a natriuretic peptide isANP, the ANP can include the sequence set forth in SEQ ID NO:1. Thecatecholamine inhibitor can include an agent that can acceleratecatecholamine degradation (e.g., a monoamine oxidase A (MAO-A) activatoror a catechol-O-methyltransferase (COMT) activator). The catecholamineinhibitor can include an agent that can block catecholamine release(e.g., gabapentin). The catecholamine inhibitor can include both anatriuretic peptide (e.g., ANP) and a hydroxylase inhibitor (e.g.,metyrosine). The catecholamine inhibitor can include an agent thatblocks an adrenergic receptor (e.g., an α1 adrenergic receptor) such asprazosin.

In another aspect, this document features a method for inhibitingcatecholamine synthesis and/or catecholamine secretion in a mammal. Themethod includes, or consists essentially of, administering acatecholamine inhibitor to the mammal. The catecholamine can beepinephrine, norepinephrine, dopamine, or any combination thereof. Forexample, the catecholamine can be epinephrine. The mammal can be ahuman. The catecholamine inhibitor can include a tyrosine hydroxylaseinhibitor (e.g., metyrosine). The catecholamine inhibitor can include anatriuretic peptide (e.g., ANP, BNP, CNP, and DNP). When a natriureticpeptide is ANP, the ANP can include the sequence set forth in SEQ IDNO:1. The catecholamine inhibitor can include an agent that canaccelerate catecholamine degradation (e.g., a MAO-A activator or a COMTactivator). The catecholamine inhibitor can include an agent that canblock catecholamine release (e.g., gabapentin). The catecholamineinhibitor can include both a natriuretic peptide (e.g., ANP) and ahydroxylase inhibitor (e.g., metyrosine). The catecholamine inhibitorcan include an agent that blocks an adrenergic receptor (e.g., an α1adrenergic receptor) such as prazosin.

In another aspect, this document features a method for preventingtransplant rejection in a mammal. The method includes, or consistsessentially of, administering a catecholamine inhibitor to the mammal.The transplant rejection can include graft-versus-host disease. Themammal can be a human. The catecholamine inhibitor can include atyrosine hydroxylase inhibitor (e.g., metyrosine). The catecholamineinhibitor can include a natriuretic peptide (e.g., ANP, BNP, CNP, andDNP). When a natriuretic peptide is ANP, the ANP can include thesequence set forth in SEQ ID NO:1. The catecholamine inhibitor caninclude an agent that can accelerate catecholamine degradation (e.g., aMAO-A activator or a COMT activator). The catecholamine inhibitor caninclude an agent that can block catecholamine release (e.g.,gabapentin). The catecholamine inhibitor can include both a natriureticpeptide (e.g., ANP) and a hydroxylase inhibitor (e.g., metyrosine). Thecatecholamine inhibitor can include an agent that blocks an adrenergicreceptor (e.g., an α1 adrenergic receptor).

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention pertains. Although methods and materialssimilar or equivalent to those described herein can be used to practicethe invention, suitable methods and materials are described below. Allpublications, patent applications, patents, and other referencesmentioned herein are incorporated by reference in their entirety. Incase of conflict, the present specification, including definitions, willcontrol. In addition, the materials, methods, and examples areillustrative only and not intended to be limiting.

The details of one or more embodiments of the invention are set forth inthe accompanying drawings and the description below. Other features,objects, and advantages of the invention will be apparent from thedescription and drawings, and from the claims.

DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1E show failure of therapeutic interventions in C. novyi-NTtherapy-induced toxicity. Mice bearing large subcutaneous CT26 tumors(600-900 mm³) were injected with 12 million parental C. novyi-NT sporesintra-tumorally along with the indicated agents. Shown are theKaplan-Meier survival curves of animals that received the antibioticmetronidazole (Figure A), dexamethasone (Figure B), or antibodies to thereceptors for the pro-inflammatory cytokines including anti-IL-6R(Figure C), anti-mIL-3 (Figure D) and anti-TNF-α (Figure E) antibodies.

FIGS. 2A-2E show ANP-C. novyi-NT. FIG. 2A shows that a series of cloneswere selected and analyzed for ANP secretion in bacterial cultures by anenzyme-linked immunosorbent assay (ELISA). Clone 1-29 had the highestlevel of ANP secretion. FIG. 2B shows that several clones of ANP-C.novyi-NT were selected for testing cGMP induction in bovine aorticendothelial cells. cGMP induction was measured by ELISA. FIG. 2C showsthat clones of ANP-C. novyi-NT showed comparable growth patternscompared to the parental C. novyi-NT. FIG. 2D shows that plasma ANPlevels of CT26 tumor-bearing mice at 36 hours after ANP-C. novyi-NTspore injection. FIG. 2E shows that peak levels of additional cytokinesat 36 hours after spore injection. All data are presented as means±SD.

FIGS. 3A-3C show that ANP reduces mortality from the cytokine releasesyndrome. FIG. 3A shows a Kaplan-Meier Curve (top panel) and therapeuticresponse (bottom panel) of ANP-C. novyi-NT compared to parental C.novyi-NT with or without supplemental ANP delivered via osmotic pumps.FIG. 3B shows haematoxylin and eosin (H&E) and anti-Ly6G antibodystained sections of the lungs, liver, and spleen. FIG. 3C shows cytokinelevels measured at 36 hours after spore injection. All data arepresented as means±SD.

FIG. 4 shows that ANP-C. novyi-NT reduces therapy-induced mortality.GL-261 glioblastoma cells were subcutaneously implanted into C57BL/6mice. Once the tumor reached 600-900 mm³, 12 million C. novyi-NT orANP-C. novyi-NT spores were directly injected into the tumor and themice were monitored for survival. Kaplan-Meier survival curves of C.novyi-NT and ANP-C. novyi-NT treated animals are shown.

FIGS. 5A-5C show that ANP prevents death from septic shock induced byCLP. FIG. 5A contains Kaplan-Meier curves showing survival of C57BL/6mice after CLP. ANP delivery via osmotic pump was initiated 12 hoursprior to CLP and was continued for 7 days. FIG. 5B contains H&E sectionsof the lungs and liver obtained 24 hours after CLP showed lower orabsent pulmonary septal thickening and vacuolization in ANP-treatedmice, indicating reduced inflammation. FIG. 5C shows cytokine andchemokine levels obtained at 24 hours after CLP. All data are presentedas means±SD.

FIGS. 6A-6B show that ANP prevents death from septic shock induced byCLP FIG. 6A contains macroscopic images of the intestines taken 24 hoursafter CLP demonstrating the cecal inflammation and necrosis (arrows).FIG. 6B shows cytokine levels obtained at 24 hours after CLP. All dataare presented as means±SD.

FIG. 7 shows that IκB kinase inhibition did not improve survival in C.novyi-NT therapy-induced sepsis. Kaplan-Meier survival curves of micetreated with IκB kinase inhibitor BMS345541 while undergoing C. novyi-NTtherapy.

FIGS. 8A-8D. Effects of the inhibition of catecholamine synthesis. FIG.8A shows peritoneal macrophages stimulated with LPS at 50 μg/ml invitro. Culture supernatants were collected after 24 hours and analyzedby ELISA for epinephrine and norepinephrine levels. LPS inducedmacrophage-derived catecholamine production, which was effectivelyblocked by pre-incubation with ANP or metyrosine 10 minutes before. FIG.8B shows cytokine levels that were measured in supernatants frommacrophage cultures treated as in (FIG. 8A). FIG. 8C shows catecholaminelevels measured in supernatants from epinephrine-stimulated peritonealmacrophages after pre-treatment with ANP or metyrosine. Epinephrine wasused at the physiological concentration of 15 ng/ml. FIG. 8D showscytokine levels measured in peritoneal macrophages treated as in (FIG.8C). All data are presented as means±SD.

FIGS. 9A-9D shows dopamine levels in the experimental models. FIG. 9Ashows dopamine levels in culture supernatants of peritoneal macrophagesexposed to LPS and epinephrine with or without pre-treatment with ANP ormetyrosine. FIG. 9B shows plasma dopamine levels in mice treated withLPS with or without pre-treatment with metyrosine. FIG. 9C shows plasmadopamine levels in CT26 tumor-bearing mice treated with the ANP-C.novyi-NT strain or parental C. novyi-NT with or without metyrosinepre-treatment. FIG. 9D shows plasma dopamine levels in mice undergoingCLP with or without metyrosine treatment. All data are presented asmeans±SD.

FIGS. 10A-10E shows that exogenous epinephrine exaggerates theinflammatory response, which can be inhibited by ANP and catecholaminesynthesis inhibitor metyrosine. FIGS. 10A and 10B show peritonealmacrophages pre-treated with ANP or metyrosine for 10 minutes, thenstimulated with epinephrine at 15 ng/ml plus LPS at 50 μg/ml. Culturesupernatants were analyzed for epinephrine and norepinephrine levels(FIG. 10A) as well as levels of the indicated cytokines and chemokines(FIG. 10B). FIG. 10C shows survival of BALB/c mice after the indicatedtreatments. FIGS. 10D and 10E show plasma catecholamine levels (FIG.10D) as well as levels of indicated cytokines and chemokines (FIG. 10E)in mice receiving LPS or LPS plus epinephrine with or without metyrosinepre-treatment. All data are presented as means±SD.

FIGS. 11A-11C show catecholamine and additional cytokine data from theCART19 experiments. FIGS. 11A and 11B show co-cultures of CART19 andRaji with or without metyrosine and ANP were stimulated with 15 ng/ml ofepinephrine in vitro. Culture supernatant were collected after 24 hoursand analyzed for catecholamines (FIG. 11A) and the indicated cytokines(FIG. 11B). Epinephrine (old): epinephrine at 15 ng/ml was incubated at37° C. for 24 hours in the cell-free medium. Epinephrine (new):epinephrine at 15 ng/ml was added into the cell-free medium andimmediately measured. FIG. 11C shows plasma dopamine levels in micecarrying Raji tumors at two time points after CART19 treatment.Metyrosine was able to reduce dopamine production.

FIGS. 12A-12F show suppression of catecholamines with metyrosine reducestoxicity from bacteria-generated sepsis. FIG. 12A shows survival of CT26tumor-bearing BALB/c mice undergoing C. novyi-NT therapy with or withoutmetyrosine pre-treatment. FIG. 12B shows plasma levels of epinephrineand norepinephrine from CT26 tumor-bearing mice treated with parental C.novyi-NT spores with or without metyrosine pre-treatment, compared toANP-C. novyi-NT-treated mice. FIG. 12C shows plasma levels of indicatedcytokines at 36 hrs after C. novyi-NT spore administration with orwithout metyrosine pre-treatment. FIG. 12D shows survival of C57BL/6mice undergoing CLP with or without metyrosine and imipenempre-treatments. FIG. 12E shows plasma levels of epinephrine andnorepinephrine at different time points after CLP with or withoutmetyrosine pre-treatment. FIG. 12F shows plasma levels of indicatedcytokines after CLP, with or without metyrosine pre-treatment. Data arepresented as means±SD.

FIGS. 13A-13G shows that inhibition of catecholamine synthesis reducesCRS after anti-CD3 and CART19 treatment. FIG. 13A shows survival of micetreated with anti-CD3 with or without metyrosine pre-treatment. FIG. 13Bshows levels of epinephrine and norepinephrine measured at 24 hoursafter anti-CD3 treatment with or without metyrosine. FIG. 13C showsplasma levels of indicated cytokines at 24 hours after anti-CD3treatment with or without metyrosine. FIG. 13D shows in vitro co-cultureof CART19 with Raji cells (5:1) increased catecholamine production(epinephrine, norepinephrine). Both metyrosine and ANP suppressed thecatecholamine surge. FIG. 13E shows that CART19-induced release ofindicated cytokines was blocked by metyrosine and ANP in vitro. FIG. 13Fshows that in vivo CART19 treatment increased circulatingcatecholamines, assessed at 24 and 72 hours after CART19 IV injection.Metyrosine was able to block that effect. FIG. 13G shows that theindicated circulating mouse and human cytokines were significantlylowered in metyrosine pre-treated mice. The data are presented as themean±SD.

FIGS. 14A-14B show dopamine and additional cytokine data from theanti-CD3 experiments. FIG. 14A shows levels of dopamine measured at 24hours after anti-CD3 treatment with or without metyrosine. FIG. 14Bshows levels of indicated cytokines measured at 24 hours after anti-CD3treatment with or without metyrosine.

FIG. 15 contains a schematic showing how inhibition of the catecholaminepathway may reduce CRS. TLR, toll-like receptor.

FIGS. 16A-16M show in vitro and in vivo studies of ANP-C. novyi-NT. FIG.16A shows Kaplan-Meier curves of mice with large subcutaneous CT26tumours (600-900 mm³), treated with intratumorally injected 12×10⁶ C.novyi-NT spores and the indicated agents: anti-IL-6R (n=10),metronidazole (n=5), dexamethasone (n=6), anti-IL3 (n=6) and anti-TNF-α(n=5) compared to controls (n=5). Survival differences were analysed bytwo-sided log-rank test. FIGS. 16B and 16C show selected clones ofANP-C. novyi-NT were analysed for ANP secretion, shown as the average ofa triplicate, (B) and for cGMP induction (n=3) using bovine aorticendothelial cells (C). FIG. 16D shows a growth pattern of several clonescompared to the parental C. novyi-NT. The average of a triplicate isshown. FIGS. 16E-16G shows levels of plasma ANP (left to right, n=7, 8,7, 5 independent samples per column) (E), plasma cGMP (n=5, 5, 4, 4samples per column) (F) and germinated C. novyi strains in tumour tissue(n=4 samples per column) based on quantification cycle (C_(q)) of RT-PCRof germination-specific NT01CX1854 gene (G), measured at 36 hours afterspore injection. FIG. 16H shows representative haematoxylin and eosin aswell as anti-CD11b antibody stained sections from the lungs, liver,spleen and bone marrow of mice treated with ANP-C. novyi-NT (n=3), C.novyi-NT (n=3) and C. novyi-NT plus ANP (n=2) compared to normalcontrols (n=2). FIGS. 16I-16M show pulmonary permeability (n=4 mice pergroup), lung wet-dry ratio (n=3 mice per group) (I) as well as levels ofcytokines (n=6 independent samples per column) (J), dopamine (n=3independent samples per column) (K), haematocrit (n=3, 5, 4, 4 samplesper column) (L) and calculated plasma volume (n=3, 5, 4, 4 samples percolumn) (M) measured 36 hours after spore treatment. Data in FIGS. 16C,16E-16G, and 16I-16M are presented as mean±s.d. with individual datapoints shown, analysed by two-tailed t-test. BAEC, bovine aorticendothelial cells.

FIGS. 17A-17D show that ANP reduces mortality. FIG. 17A shows aKapla-Meier curve (top panel) and therapeutic response (bottom panel) ofANP-C. novyi-NT (n=16) compared to C. novyi-NT (n=16), C. novyi-NT withANP via osmotic pump (n=12) and vector C. novyi-NT control (n=5).Statistical survival differences were evaluated by two-sided log-ranktest. FIG. 17B shows representative anti-CD11b-antibody-stained sectionsfrom the lungs, liver, spleen and bone marrow of mice treated withANP-C. novyi-NT (n=3) and C. novyi-NT (n=3) compared to normal controls(n=2). FIG. 17C shows plasma levels of indicated cytokines (n=6independent samples per group) 36 hours after spore injection. FIG. 17Dshows corresponding plasma levels of epinephrine and norepinephrine 36hours after C. novyi-NT, ANP-C. novyi-NT and C. novyi-NT plus ANP pumpcompared to normal controls (n=3 per group). FIG. 17C and FIG. 17D dataare presented as mean±s.d. with individual data points shown, analysedby two-tailed t-test.

FIGS. 18A and 18B show survival of mice treated with ANP and IκB kinaseinhibitor BMS345541. FIG. 18A shows survival of mice with subcutaneouslyimplanted GL-261 tumours, treated with 12×10⁶ of ANP-C. novyi-NT spores(n=10 animals per group). FIG. 18B shows survival of mice treated withC. novyi-NT and IκB kinase inhibitor BMS345541 (n=5 mice per group).Survival differences were analysed by two-sided log-rank test.

FIGS. 19A-19D show that epinephrine increases catecholamine levels andenhances the inflammatory response. FIG. 19A shows survival of BALB/cmice implanted with the indicated catecholamine pump and stimulated witha sublethal dose of LPS (n=14 mice per group) compared to LPS alone(n=19 mice). Survival differences were analysed byGehan-Breslow-Wilcoxon test. FIG. 19B shows survival of BALB/c mice withindicated catecholamine pump without LPS stimulation (n=5 mice pergroup). FIGS. 19C and 19D shows 24 hour plasma levels of epinephrine(left to right, n=3, 4, 3, 3, 3, 4, 4, 3 per column), norepinephrine(n=3, 3, 3, 3, 3, 4, 4, 3) and dopamine (n=3, 3, 3, 3, 3, 4, 4, 3) (C)as well as levels of IL-6 (n=4 per column), TNF-α (n=5 per column) andKC (n=4 per column) (D) in mice receiving the indicated treatments. FIG.19E shows dopamine concentration of LPS and epinephrine treatedperitoneal macrophages pre-incubated with ANP or MTR (n=3 per column),measured after 24 hours. FIGS. 19F and 19G show levels of catecholamines(n=3 independent samples per column) (F) and several cytokines (n=3independent samples per column) (G) in epinephrine (15 ng ml⁻¹)-treatedperitoneal macrophages pre-incubated with ANP or MTR and measured after24 hours. Data in FIGS. 19C-19G are presented as mean±s.d. withindividual data points shown, analysed by two-tailed t-test.

FIGS. 20A-20B show that catecholamine production in myeloid cells isessential for cytokine release. FIG. 20A shows peritoneal macrophagesthat were pre-incubated with ANP or MTR for 10 minutes and thenstimulated with LPS (50 μg ml⁻¹) or a combination of LPS and epinephrine(15 ng ml⁻¹) in vitro. Shown are the levels of epinephrine (left toright, n=3, 3, 3, 6, 6, 6, 3, 3, 3 per column) and norepinephrine (n=3)in the supernatant after 24 hours. FIG. 20B shows correspondingcytokines from macrophage culture supernatants: IL-6 (n=3, 3, 3, 4, 4,4, 3, 3, 3), MIP-2 (n=4, 4, 4, 4, 5, 5, 4, 3, 3), KC (n=3, 3, 3, 5, 5,5, 3, 3, 3) and TNF-□ (n=3, 3, 3, 3, 5, 6, 4, 3, 3). Data in FIGS.20A-20B are presented as mean±s.d. with individual data points shown,analysed by two-tailed t-test.

FIGS. 20C-20E shows that catecholamines derived from myeloid cellsmodulate the cytokine release in vivo. FIG. 20C shows survival ofTh^(+/+) and Th^(ΔLysM) mice treated with LPS and analysed withtwo-sided log-rank test (n=12; 6 male, 6 female). FIGS. 20D and 20E showplasma levels of epinephrine (n=4, 4, 7, 6) and norepinephrine (n=3, 3,7, 6) (D) and indicated cytokines (n=3, 3, 4, 3) (E) at baseline and 24hours after LPS treatment in Th^(+/+) or Th^(ΔLysM) mice. Data in FIGS.20D and 20E are presented as mean±s.d. with individual data pointsshown, analysed by two-tailed t-test.

FIGS. 21A-21E shows autocrine and LPS-induced catecholamine productionenhance the cytokine release in human U937 macrophage line. FIGS. 21Aand 21B show U937 cells that were pre-treated with ANP or MTR for 10minutes, then stimulated with LPS at 1 μg ml⁻¹ and/or epinephrine at 15ng ml⁻¹. Culture supernatants were analysed for catecholamines (n=3 percolumn) (A) as well as the indicated cytokines (n=3 per column) (B).FIG. 21C shows TH expression of baseline and LPS-stimulated Th^(+/+) orTh^(ΔLysM) macrophages (n=3 per group), analysed by qPCR; results arenormalized by ubiquitin C (UBC). FIGS. 21D and 21E show supernatants ofcollected peritoneal macrophages from Th^(+/+) or Th^(ΔLysM) mice,stimulated with LPS at 50 μg ml⁻¹, epinephrine 15 μg ml⁻¹ or both for 24hours, were analysed for levels of epinephrine (n=3), norepinephrine(n=3) (D) and cytokines IL-6 (n=3), KC (n=3), MIP-2 (n=3) and TNF-α(n=3) (E). All data are presented as mean±s.d. with individual datapoints shown, analysed by two-tailed t-test.

FIGS. 22A-22E show metyrosine (MTR) dose-dependently improves survivaland cytokine release. FIG. 22A shows survival of BALB/c mice stimulatedwith a lethal dose of LPS and treated with the indicated dose of MTR:MTR 20 mg kg⁻¹ (n=5 mice per group); MTR 30 mg kg⁻¹ (n=10 mice), MTR 40mg kg⁻¹ (n=12) compared to LPS (n=10 mice). Survival differences wereanalysed by two-sided log-rank test. FIGS. 22B and 22C show levels ofplasma catecholamines (n=4 per column) (B) and IL-6 (n=4 per column), KC(left to right, n=4, 4, 3 per column), IFN-γ (n=4) and TNF-α (n=4, 4, 3)(C) at different MTR doses measured 24 hour after LPS injection. FIGS.22D and 22E shows 24-hour-time courses of circulating epinephrine (n=5,5, 5, 4, 5, 4, 5, 5), norepinephrine (n=5) and dopamine (n=5) (D) andcorresponding levels of IL-6 (n=4), KC (n=7, 7, 7, 6, 5, 4, 5, 5), IFN-γ(n=6, 6, 6, 8, 4, 8, 6, 4) and TNF-α (n=6, 6, 6, 6, 6, 4, 7, 7) (E) inLPS-treated mice receiving MTR 40 mg kg⁻¹. Data in FIGS. 22B-22E arepresented as mean±s.d. with individual data points shown, analysed bytwo-tailed t-test.

FIGS. 23A-23H shows that suppression of catecholamines with metyrosinereduces toxicity of oncolytic bacterium C. novyi-NT and polymicrobialsepsis. FIG. 23A shows survival (top panel) and therapeutic response(bottom panel) of CT26 tumour-bearing BALB/c mice undergoing C. novyi-NTtreatment with or without MTR pre-treatment (n=13 mice per group).Survival differences were analysed with two-sided log-rank test. FIGS.23B and 23C show corresponding plasma levels of epinephrine (n=3independent samples per column), norepinephrine (n=3), dopamine (n=3)(B) and indicated cytokines (left to right, n=3, 3, 6, 7 independentsamples per column) (C), measured at baseline and 36 hours aftertreatment. FIG. 23D shows survival of C57BL/6 mice undergoing CLP, withthe indicated treatments (CLP, n=20 mice; MTR, n=22; IMP, n=19; MTR+IMP,n=20 mice per group). Survival differences were analysed with two-sidedlog-rank test. FIG. 23E shows plasma levels of epinephrine (n=3),norepinephrine (n=3) and dopamine (n=3) at the indicated time pointsafter CLP, with or without MTR pre-treatment. FIG. 23F shows levels ofindicated cytokines (n=3) at baseline and 24 hours after CLP, with orwithout MTR pre-treatment. FIGS. 23G and 23H show levels of plasmadopamine (left to right, n=3, 8, 8 independent samples per column) (G)and KC (n=6, 6, 6, 5), IL-2 (n=6, 6, 6, 5) and IFN-γ (n=6) (H) measured24 hours after α-CD3 treatment, with or without MTR. Data in FIGS. 23B,23C, and 23E-23H are presented as mean±s.d. with individual data pointsshown, analysed by two-tailed t-test.

FIGS. 24A-24C shows blockage of α1-adrenoceptor mediates the survival inexperimental systemic inflammatory syndrome. FIG. 24A shows Kaplan-Meiercurve of LPS-injected BALB/c mice treated with the indicatedadrenoreceptor blockers (n=15 animals per group). FIGS. 24B and 24C show1Levels of epinephrine, norepinephrine (left to right, n=3, 5, 8 percolumn) and dopamine (n=5, 4, 7) (B) as well as indicated cytokines(n=3, 5, 8) (C) measured 24 hours after LPS administration. Data inFIGS. 24B and 24C are presented as mean±s.d. with individual data pointsshown, analysed by two-tailed t-test.

FIGS. 25A-25E show inhibition of catecholamine synthesis reduces CRSafter anti-CD3 treatment. FIGS. 25A and 25B show levels of epinephrineand norepinephrine (left to right, n=3, 3, 8, 8 independent samples percolumn) (A) and of cytokines (n=6 independent samples) (B) measured 24hours after anti-CD3 treatment, with or without MTR. FIG. 25C showssurvival of BALB/c mice treated with anti-CD3, with or without MTR (n=15animals); analysed by two-sided log-rank test. FIGS. 25D and 25E showlevels of epinephrine, norepinephrine (n=3, 3, 4, 4) (D) and indicatedcytokines (n=3, 3, 4, 4) (E) measured 24 hours after anti-CD3 treatmentin Th^(+/+) or Th^(ΔLysM) mice. Data in FIGS. 25A, 25B, 25D, and 25E arepresented as mean±s.d. with individual data points shown, analysed bytwo-tailed t-test.

FIGS. 26A-26E show catecholamine and additional cytokine data from thehCART19 in vitro experiments. FIG. 26A shows levels of catecholamines inRaji cells (n=3), hCART19 (n=3) and UT-T (n=3 per column) at baselineand when exposed to epinephrine. FIGS. 26B and 26C show co-cultures ofhCART19 and Raji with or without MTR or ANP pre-treatment werestimulated with 15 ng ml⁻¹ of epinephrine in vitro. Culture supernatantswere collected after 24 hours and analysed for epinephrine (left toright, n=4, 4, 4, 3, 3, 3, 2, 2 per column) and norepinephrine (n=4, 4,3, 3, 3, 3, 2, 2). Epinephrine (old): epinephrine at 15 ng ml⁻¹ wasincubated at 37° C. for 24 hours in the cell-free medium. Epinephrine(new): epinephrine at 15 ng ml⁻¹ was added into the cell-free medium andimmediately measured (B). Corresponding cytokine levels of MIP-1α (n=4,4, 3, 4, 3, 3, 3, 3, 3), IFN-γ (n=4, 4, 3, 4, 3, 4, 4, 4, 4), IL-2 (n=4,4, 3, 4, 3, 3, 3, 3, 3) and TNF-α(n=4, 4, 3, 4, 3, 3, 3, 3, 3) (C). UT-Tserved as control. FIGS. 26D and 26E show co-cultures of hCART19 andRaji with or without CHX were stimulated with 15 ng ml⁻¹ of epinephrinein vitro. Levels of catecholamines (n=3, 3, 3, 3, 3, 3, 3, 3, 3, 3, 3,3, 1, 1) (D) and indicated human cytokines (n=3) (E) were measured after24 hours. Data are presented as mean±s.d. with individual data pointsshown, analysed by two-tailed t-test.

FIGS. 27A-27E show inhibition of catecholamine production reducescytokine release from activated hCART19. FIGS. 27A and 27B show levelsof epinephrine (left to right, n=4, 4, 4, 3, 3, 3 per column) andnorepinephrine (n=4, 4, 3, 3, 3, 3) (A) and corresponding cytokinesMIP-1α (n=3), TNF-α (n=4, 4, 4, 3, 3, 3), IFN-γ (n=4, 4, 4, 3, 3, 3) andIL-2 (n=4, 3, 3, 3, 3, 3) (B) in the supernatant 24 hours afterincubation of Raji cells with hCART19 or UT-T (ratio 1:5), with orwithout MTR or ANP. FIG. 27C shows Kaplan Meier curves showing thesurvival of Raji-bearing NSGS mice with high tumour burden, treated with1.5×10⁷ hCART19, with or without MTR pre-treatment compared to UT-T, MTRand no treatment (n=5 mice per group). Survival differences wereevaluated by two-sided log-rank test. FIG. 27D and FIG. 27E shows levelsof circulating epinephrine and norepinephrine (n=3, 3, 5, 4, 4, 5, 4, 5,7, 8) (D) and of indicated circulating mouse and human cytokines (n=4samples per group) (E), assessed 24 and 72 hours after administration ofhCART19 with or without MTR in comparison to controls. Data in FIGS.27A, 27B, 27D, and 27E are presented as mean±s.d. with individual datapoints shown, analysed by two-tailed t-test.

FIGS. 28A-28I show that MTR and ANP prevent cytokine release inRaji/hCART19 mouse model. FIG. 28A shows representative bioluminescentimages (BLI) of Raji-bearing NSGS mice with high tumour burden. At day0, the tumour engraftment was quantified by BLI and mice were randomlyassigned to the respective treatment groups (n=5 mice per group). FIGS.28B and 28C show levels of dopamine (left to right, n=3, 3, 3, 4, 4, 4,3, 4, 4, 4 per column) (B) and indicated cytokines (n=4) (C) measured inmice (with high tumour burden) 24 and 72 hours after hCART19 and UT-Tadministration. FIG. 28D shows representative BLI of Raji-bearing NSGSmice with low tumour burden. At day 0, mice were randomly assigned basedon tumour burden to receive hCART19, with or without MTR (n=10 mice pergroup) or UT-T, with or without MTR (n=5 mice per group). FIG. 28E showslevels of human hIL-2 (n=4, 4, 3) and mMIP-2 (n=3, 3, 4) assessed 72hours after hCART19 injection in mice with low tumour burden. FIGS. 28Fand 28G show NSGS mice that were injected with hCART19 4 days after Rajiimplantation and treated with ANP delivered via subcutaneously implantedosmotic pumps. Levels of circulating catecholamines (n=4 per column) (F)and mIL-6, mKC and mMIP-2 (n=4, 4, 3, 4) as well as hIL-2 (n=4) (G) wereassessed 24 hours after hCART19 administration. FIG. 28H shows survivalof Raji cell-bearing NSGS mice treated with hCART19 and ANP (n=5 pergroup); analysed by two-sided log-rank test. FIG. 28I shows level ofcirculating hCART19 10 days after treatment, determined by C_(q) by qPCRand analysed in triplicates (n=4 per group). Data in FIGS. 28B, 28C, and28E-28I are presented as mean±s.d. with individual data points shown,analysed by two-tailed t-test.

FIGS. 29A-29D show inhibition of catecholamine synthesis with metyrosinedoes not impair the therapeutic response of hCART19. FIG. 29A showsserial bioluminescence imaging (BLI) of Raji-bearing NSGS mice (lowtumor burden) at day 6 and 19 after treatment with 1.5×10⁷ hCART19, withor without MTR (n=10 mice per group) compared to control (UT-T), with orwithout MTR (n=5 mice per group). BLI counts were used to quantify thetumour burden during the treatment course (right). Statisticaldifferences were evaluated by one-tailed t-test. FIG. 29B showsCorresponding Kaplan-Meier curve of Raji-bearing NSGS mice with lowtumour burden, treated with 1.5×10⁷ hCART19, with or without MTRpre-treatment (n=10 mice per group) in comparison to control (UT-T),with or without MTR (n=5 mice per group). Survival differences wereanalysed by weighted log-rank test. FIGS. 29C and 29D show Levels ofplasma epinephrine (n=3, 3, 4 per column) and norepinephrine (n=3, 4, 7)(C) and human hIFN-γ (n=4), hTNF-α (n=4, 3, 3), and mouse cytokinesmIL-6 (n=3) and KC (n=3) (D), assessed 72 hours after hCART19 treatment.Data are presented as mean±s.d. with individual data points shown,analysed by two-tailed t-test.

FIGS. 30A-30D show metyrosine and ANP prevent cytokine release insyngeneic Eη-ALL model without compromising antitumor efficacy. FIG. 30Aand FIG. 30B show circulating catecholamines (left to right, n=3, 4, 3,4, 4, 4, 3, 4, 3, 4, 4, 4 per column/graph) (A) and murine cytokinesIL-6 (n=3 per column), KC (n=3, 3, 3, 4, 3, 3, 4, 4, 3 per column),IL-1α (n=3, 3, 3, 4, 3, 3, 4, 3, 3 per column) and GCSF (n=3, 3, 3, 4,4, 3, 4, 3, 3 per column) (B), assessed at 24 and 72 hours after mCART19injection. Data are presented as means±s.d. with individual data pointsshown, analysed by two-tailed t-test. FIG. 30C shows BLI performedbefore and 10 days after mCART19 cell injection, with or without ANP andMTR pre-treatment (n=5 animals per group). Quantification of BLIradiance was used as a surrogate measurement of tumour burden during thetreatment course (right). FIG. 30D shows percentage survival ofEϵ-ALL-mice after mCART19 cell transfer (n=8 mice per group). Survivaldifferences were analysed by two-sided log-rank test.

DETAILED DESCRIPTION

This document provides methods and materials for treating and/orpreventing CRS. For example, this document provides methods andmaterials for using one or more catecholamine inhibitors to treat amammal having CRS. For example, this document provides methods andmaterials for using one or more catecholamine inhibitors to prevent CRSin a mammal at risk of developing CRS. As used herein, a “catecholamineinhibitor” can be any agent that can disrupt a catecholamine responseloop (see, e.g., FIG. 15). For example, a catecholamine inhibitor can bean agent capable of suppressing catecholamine synthesis. For example, acatecholamine inhibitor can be an agent capable of blocking anadrenergic receptor. Examples of agents that can be used to disrupt thecatecholamine synthesis loop include, without limitation, natriureticpeptides, tyrosine hydroxylase inhibitors (e.g., metyrosine), agentsthat accelerate catecholamine degradation, agents that blockcatecholamine release, agents that block adrenergic receptors (e.g.,prazosin), and any other agents that interrupt this catecholamineresponse loop by unknown mechanisms.

In some cases, one or more catecholamine inhibitors described herein(e.g., natriuretic peptides, tyrosine hydroxylase inhibitors, and/oragents that blocks adrenergic receptors (e.g., an α1 adrenergicreceptor)) can be used to reduce and/or eliminate cytokine and/orchemokine release. A cytokine and/or chemokine can be a pro-inflammatorycytokine. Examples of cytokines and chemokines include, withoutlimitation, tumor necrosis factor-alpha (TNF-α), interleukin 1 beta(IL-1(3), interleukin 6 (IL-6), interleukin 10 (IL-10), interleukin 1receptor antagonist (IL-1RA), interferon gamma (IFNγ), CXCL1 (KC),macrophage inflammatory protein 2 (MIP-2), macrophage inflammatoryprotein 1 beta (MIP-1β), and granulocyte-colony stimulating factor(G-CSF). For example, the methods and materials provided herein can beused to reduce and/or eliminate production of IL-6, IFNγ, TNF-α, KC,MIP-2, and MIP-1β.

In some cases, one or more catecholamine inhibitors described herein(e.g., natriuretic peptides, tyrosine hydroxylase inhibitors, and/oragents that blocks adrenergic receptors (e.g., an α1 adrenergicreceptor)) can be used to reduce and/or eliminate cytokine and/orchemokine release from any appropriate type of cell. A cell can be an invivo cell. A cell can be an in vitro cell. Examples of cell typesinclude, without limitation, myeloid cells (e.g., activated myeloidcells), granulocytes, monocytes, T cells (e.g., activated T cells), andmacrophages.

In some cases, one or more catecholamine inhibitors described herein(e.g., natriuretic peptides, tyrosine hydroxylase inhibitors, and/oragents that blocks adrenergic receptors (e.g., an α1 adrenergicreceptor)) can be used to reduce and/or eliminate catecholaminesynthesis. Examples of catecholamines include, without limitation,epinephrine (EPI), norepinephrine (NE), and L-Dopamine (DOP). Forexample, the methods and materials provided herein can be used toinhibit EPI synthesis.

When treating and/or preventing CRS as described herein, the CRS can beany appropriate type of CRS. In some cases, CRS can be associated withan infection. Examples of CRS-associated infections include, withoutlimitation, bacterial infections (e.g., gram-positive bacterialinfections and gram-negative bacterial infections), polymicrobialinfections, viral infections (e.g., Ebola infections, avian influenzainfections, and smallpox infections. In some cases, CRS can beassociated with administration of an immunotherapy. Immunotherapy can bea cancer immunotherapy. Examples of immunotherapies include, withoutlimitation, antibody therapies (e.g., orthoclone OKT3, muromonab-CD3,rituximab, alemtuzumab, ipilimumab, nivolumab, ofatumumab, CP-870,893,LO-CD2a/BTI-322, or TGN1412), chimeric antigen receptor therapies(CAR-T; e.g., tisagenlecleucel or axicabtagene ciloleucel), bi-specificT-cell engagers (BiTEs), cellular immunotherapies (e.g., adoptive T-celltherapy or dendritic cell therapy), cytokine therapies (e.g., interferontherapy and interleukin therapy), and microorganism therapies (e.g.,bacterial therapy or viral therapy). In cases where CRS is associatedwith an immunotherapy, and the immunotherapy is CAR-T, the CAR-T cantarget any of a variety of antigens (e.g., CD19, CD20, CD22, CD30, CEA,EGFR, EGP-2, EGP-40, erb-B2 (also referred to as Her2/neu), FBP, fetalacetylcholine receptor, GD2, GD3, IL-13R-a2, KDR, k-light chain, LeY,MAGE-A1, MUC1, NKG2D ligands, oncofetal antigen (h5T4), PSCA, PSMA,TAG-72, and VEGF-R2). In cases where CRS is associated with animmunotherapy, and the immunotherapy is CAR-T, the CAR-T can be asdescribed elsewhere (see, e.g., Ruella et al., 2016 Curr Hematol MaligRep., 11:368-84). In cases where CRS is associated with microorganismtherapy, the microorganism therapy can use live microorganisms,attenuated microorganisms, inactivated microorganisms, or anycombination thereof. In some cases, CRS can be associated with atreatment (e.g., an immunotherapeutic agent) for an autoimmune disease.Examples of autoimmune diseases include, without limitation, rheumatoidarthritis (RA), juvenile idiopathic arthritis (JIA), ankylosingspondylitis, psoriasis, systemic lupus erythematosus (SLE), celiacdisease, type 1 diabetes, autoimmune encephalomyelitis, multiplesclerosis, central nervous system (CNS) autoimmune demyelinatingdiseases, chronic inflammatory demyelinating polyneuropathy (CIDP),transverse myelitis, polymyositis, dermatomyositis, inflammatory boweldisease (e.g. Crohn's disease and ulcerative colitis), autoimmunehemolytic anemia, autoimmune cardiomyopathy, autoimmune thyroiditis,Graves' disease, Sjogren's syndrome, Goodpasture syndrome, autoimmunepancreatitis, Addison's disease, alopecia, myasthenia gravis,sarcoidosis, scleroderma, pemphigus vulgaris, mixed connective tissuedisease, bullous pemphigoid, and vitiligo. In some cases, CRS can beassociated with transplant rejection (e.g., organ rejection, allograftrejection, host-versus-graft disease, and graft-versus-host disease(GVHD)).

In cases where CRS is associated with transplant rejection, the methodsand materials provided herein can be used to treat and/or preventtransplant rejection. For example, one or more catecholamine inhibitorsdescribed herein (e.g., natriuretic peptides, tyrosine hydroxylaseinhibitors, and/or agents that blocks adrenergic receptors (e.g., an α1adrenergic receptor)) can be used to treat and/or prevent transplantrejection. When treating and/or preventing transplant rejection asdescribed herein, the transplant can be any appropriate transplant(e.g., organ (e.g., heart, lung, kidney, and liver) transplants, tissue(e.g., skin, cornea, and blood vessels) transplants, and cell (e.g.,bone marrow and blood) transplants). A transplant can include anallograft. A transplant can include a xenograft. Transplant rejectioncan be chronic or acute. Examples of types of transplant rejectioninclude, without limitation, organ rejection, allograft rejection,host-versus-graft disease, and GVHD. For example, the methods andmaterials provided herein can be used to treat and/or prevent GVHD.

Any type of mammal having CRS or at risk for developing CRS can betreated as described herein. Examples of mammals that can be treatedwith one or more catecholamine inhibitors described herein (e.g.,natriuretic peptides, tyrosine hydroxylase inhibitors and/or agents thatblocks adrenergic receptors (e.g., an α1 adrenergic receptor)) include,without limitation, humans, non-human primates (e.g., monkeys), dogs,cats, horses, cows, pigs, sheep, rabbits, mice, and rats. For example,humans having CRS or at risk of developing CRS can be treated with oneor more catecholamine inhibitors as described herein.

In some cases, the methods provided herein can include identifying amammal as having CRS. Any appropriate method can be used to identify amammal having CRS. For example, detection of elevated levels ofcytokines (e.g., IL-6, IFNγ, TNF-α, KC, MIP-2, and/or MIP-1β) can beused to identify a human or other mammal having CRS.

In some cases, the methods provided herein also can include assessing amammal for risk of developing CRS. Any appropriate method can be used toidentify a mammal for risk of developing CRS. For example, detection ofelevated levels of catecholamines (e.g., EPI, NE, and DPO) can be usedto identify a human or other mammal for risk of developing CRS. In somecases, increased levels of EPI (e.g., in a mammal's serum) can indicatethat a mammal is at increased risk of developing CRS. For example, amammal undergoing or scheduled to undergo immunotherapy can be at riskof developing CRS.

In some cases, a mammal can be identified as being at risk of developingCRS and can be selected for treatment as described herein. For example,a mammal identified as being at risk of developing CRS can be selectedfor treatment with one or more catecholamine inhibitors described herein(e.g., natriuretic peptides, tyrosine hydroxylase inhibitors, and/oragents that blocks adrenergic receptors (e.g., an α1 adrenergicreceptor)).

Once identified as having CRS or as being at risk for developing CRS, amammal can be administered or instructed to self-administer one or more(e.g., one, two, three, four, five, or more) catecholamine inhibitorsdescribed herein (e.g., natriuretic peptides, tyrosine hydroxylaseinhibitors, and/or agents that blocks adrenergic receptors (e.g., an α1adrenergic receptor)). In some cases, a mammal can be identified asbeing at risk of developing CRS, can be selected for treatment asdescribed herein, and one or more catecholamine inhibitors can beadministered to the mammal to treat the mammal.

A catecholamine inhibitor can be any appropriate catecholamineinhibitor. Examples of catecholamine inhibitors include, withoutlimitation, reserpine, tyramine, octopamine, guanethidine, guanadrel,amphetamine, ephedrine, pseudoepherine, phenylpropanolamine,methylphenidate, cocaine, tricyclic antidepressants, phenelzine,ipraniazide, tranylcyproamine, clorgyline-befloxatone, and selegiline.

In some cases, a catecholamine inhibitor can be a natriuretic peptide. Anatriuretic peptide can be any appropriate natriuretic peptide. Examplesof natriuretic peptides include, without limitation, atrial natriureticpeptide (ANP), brain natriuretic peptide (BNP), C-type natriureticpeptide (CNP), and dendroaspis natriuretic peptide (DNP). For example, anatriuretic peptide can be ANP. ANP can be a human ANP. In some cases, anatriuretic peptide can be administered as a mature natriuretic peptidepolypeptide. In some cases, a natriuretic peptide can be administered asa precursor peptide (e.g., prepro-ANP). An exemplary human ANPpolypeptide can include the amino acid sequenceSLRRSSCFGGRMDRIGAQSGLGCNSFRY (SEQ ID NO:1). A natriuretic peptide caninclude a peptide ring (e.g., a 17-amino acid peptide ring) formed by adisulfide bond between two cysteine residues within the natriureticpeptide amino acid sequence (e.g., at cysteine residues positions 7 and23 of SEQ ID NO:1). A natriuretic peptide can bind to one or morenatriuretic peptide receptors. Examples of natriuretic peptide receptorsinclude, without limitation, guanylyl cyclase-A (GC-A; also known asnatriuretic peptide receptor-A (NPRA/ANPA) or NPR1), guanylyl cyclase-B(GC-B; also known as natriuretic peptide receptor-B (NPRB/ANPB) orNPR2), and natriuretic peptide clearance receptor (NPRC/ANPC) or NPR3).In some cases, a human ANP polypeptide can have a sequence that deviatesfrom the ANP polypeptide sequence set forth in SEQ ID NO:1, sometimesreferred to as a variant sequence, provided the ANP polypeptidemaintains its structure (e.g., a peptide ring formed by a disulfide bondbetween two cysteine residues) and function (e.g., binding to one ormore atrial natriuretic peptide receptors. For example, an ANPpolypeptide can have at least 80 (e.g., at least 85, at least 90, atleast 95, at least 98, or at least 99) percent sequence identity to SEQID NO:1 (e.g., while maintaining the cysteine residues positions 7 and23 of SEQ ID NO:1). For example, an ANP polypeptide can have one or more(e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10) amino acid modifications (e.g.,substitutions) relative to SEQ ID NO:1. In some cases, a natriureticpeptide can be administered as a nucleic acid (e.g., cDNA) encoding anatriuretic peptide polypeptide. An exemplary human ANP nucleic acid(e.g., a coding sequence or a cDNA) can include the nucleic acidsequence TCATTAAGAAGATCTTCATGTTTTGGAGGAAGAATGGATAGAATAGGAGCTCAATCAGGATTAGGATGTAATTCATTCAGATATTAA (SEQ ID NO:2). A human ANP nucleicacid can have a sequence that deviates from the ANP nucleic acidsequence set forth in SEQ ID NO:2, sometimes referred to as a variantsequence, provided the ANP nucleic acid encodes an ANP polypeptide. AnANP nucleic acid can have at least 80 (e.g., at least 85, at least 90,at least 95, at least 98, or at least 99) percent sequence identity toSEQ ID NO:2. An ANP nucleic acid can have one or more (e.g., 2, 3, 4, 5,6, 7, 8, 9, or 10) nucleotide modifications (e.g., substitutions)relative to SEQ ID NO:2.

In some cases, a catecholamine inhibitor can be a tyrosine hydroxylaseinhibitor. A tyrosine hydroxylase inhibitor can be any appropriatetyrosine hydroxylase inhibitor. A tyrosine hydroxylase inhibitor can bean inhibitor of tyrosine hydroxylase polypeptide expression or aninhibitor of tyrosine hydroxylase polypeptide activity. Examples ofcompounds that reduce tyrosine hydroxylase polypeptide activity include,without limitation, metyrosine (also known as methyltyrosine and/ormetirosine (MTR); e.g., α-MTR), alpha-methyl-p-tyrosine (AMPT),aquayamycin, bulbocapnine, 2-hydroxyestradiol, 2-hydroxyestrone,3-iodotyrosine, and oudenone. Examples of compounds that reduce tyrosinehydroxylase polypeptide expression include, without limitation, nucleicacid molecules designed to induce RNA interference (e.g., a siRNAmolecule or a shRNA molecule), antisense molecules, and miRNAs. Forexample, a tyrosine hydroxylase inhibitor can be MTR.

In some cases, a catecholamine inhibitor can accelerate catecholaminedegradation. Examples of agents that can accelerate catecholaminedegradation include, without limitation, monoamine oxidases (MAOs; e.g.,MAO-A and MAO-B), MAO activators (e.g., glucocorticoids),catechol-O-methyltransferases (COMTs), and COMT activators. Additionalexamples of agents that can accelerate catecholamine degradation can beas described elsewhere (see, e.g., Camell et al., 2017 Nature,550:119-123).

In some cases, a catecholamine inhibitor can block the release ofcatecholamines (e.g., from cells that produce catecholamines). Examplesof agents that can block catecholamine release include, withoutlimitation, gabapentin (see, e.g., Todd et al., 2012 Anesthesiology.116:1013-1024).

In some cases, a catecholamine inhibitor can block adrenergic receptors(e.g., adrenoceptors). An adrenergic receptor can be any appropriatetype of adrenergic receptor (e.g., an alpha (α) 1, α2, beta (β) 1, or β2adrenergic receptor). Examples of agents that can block adrenergicreceptors include, without limitation, alpha-1 blockers (e.g.,acepromazine, alfuzosin, doxazosin, phenoxybenzamine, phentolamine,prazosin, tamsulosin, terazosin, and trazodone), alpha-2 blockers (e.g.,phentolamine, yohimbine, idazoxan, atipamezole, and trazodone), and betablockers (e.g., propranolol, atenolol, metoprolol, bisoprolol, timolol,nebivolol, vortioxetine, butoxamine, ICI-118,551, and SR 59230A). Insome cases, a catecholamine inhibitor can block an α1 adrenergicreceptor. Additional examples of agents that can block adrenergicreceptors can be as described elsewhere (see, e.g., Sigola et al., 2000Immunology, 100:359-63).

In some cases, a catecholamine inhibitor can include both a natriureticpeptide (e.g., ANP) and a tyrosine hydroxylase inhibitor (e.g., MTR).For example, a catecholamine inhibitor can include ANP and MTR. In somecases, a catecholamine inhibitor can include both a natriuretic peptide(e.g., ANP) and an agent that blocks an adrenergic receptor (e.g., an α1adrenergic receptor, e.g., prazosin). For example, a catecholamineinhibitor can include ANP and prazosin. In some cases, a catecholamineinhibitor can include both a tyrosine hydroxylase inhibitor (e.g., MTR)and an agent that blocks an adrenergic receptor (e.g., an α1 adrenergicreceptor, e.g., prazosin). For example, a catecholamine inhibitor caninclude MTR and prazosin. In some cases, a catecholamine inhibitor caninclude a natriuretic peptide (e.g., ANP), a tyrosine hydroxylaseinhibitor (e.g., MTR), and an agent that blocks an adrenergic receptor(e.g., an α1 adrenergic receptor, e.g., prazosin). For example, acatecholamine inhibitor can include ANP, MTR, and prazosin.

One or more catecholamine inhibitors described herein (e.g., natriureticpeptides, tyrosine hydroxylase inhibitors, and/or agents that blocksadrenergic receptors (e.g., an α1 adrenergic receptor)) can beformulated into a composition (e.g., a pharmaceutically acceptablecomposition) for administration to a mammal having CRS or as being atrisk for developing CRS. For example, a therapeutically effective amountof one or more catecholamine inhibitors described herein can beformulated together with one or more pharmaceutically acceptablecarriers (additives) and/or diluents. A pharmaceutical composition canbe formulated for administration in solid or liquid form including,without limitation, sterile solutions, suspensions, sustained-releaseformulations, tablets, capsules, pills, powders, and granules.

A composition (e.g., a pharmaceutically acceptable composition)including one or more catecholamine inhibitors described herein (e.g.,natriuretic peptides, tyrosine hydroxylase inhibitors, and/or agentsthat blocks adrenergic receptors (e.g., an α1 adrenergic receptor)) canbe administered locally or systemically. A composition containing one ormore catecholamine inhibitors described herein can be designed for oral,parenteral (including subcutaneous, intramuscular, intravenous, andintradermal), or inhaled administration. For example, a compositioncontaining one or more catecholamine inhibitors described herein can beadministered systemically by an oral administration to or inhalation bya mammal (e.g., a human). When being administered orally, a compositioncontaining one or more catecholamine inhibitors described herein can bein the form of a pill, tablet, or capsule.

One or more catecholamine inhibitors described herein (e.g., natriureticpeptides, tyrosine hydroxylase inhibitors, and/or agents that blocksadrenergic receptors (e.g., an α1 adrenergic receptor)) can beadministered to a mammal having CRS or as being at risk for developingCRS as a combination therapy with one or more additionalagents/therapies used to treat CRS. For example, a combination therapycan include administering to the mammal (e.g., a human) one or morecatecholamine inhibitors described herein together with one or more CRStreatments such antibiotics (e.g., metronidazole and dexamethasone),anti-histamines (e.g., chlorphenamine), corticosteroids (e.g.,hydrocortisone), fever reducers (e.g., acetaminophen), hydration, and/orcorrecting overhydration (e.g., by dialysis or with furosemide (e.g.,intravenous furosemide)). In cases where one or more therapeutic agentsdescribed herein are used in combination with one or more additionalagents/therapies used to treat CRS, the one or more additionalagents/therapies used to treat CRS can be administered at the same timeor independently. For example, the composition including one or moretherapeutic agents can be administered first, and the one or moreadditional agents/therapies used to treat CRS administered second, orvice versa.

The invention will be further described in the following examples, whichdo not limit the scope of the invention described in the claims.

EXAMPLES Example 1 Preventing Mortality from Therapy-Induced CytokineRelease Syndrome Materials and Methods Mice

All animal works were performed in accordance to the protocol of JohnsHopkins Animal Care and Use Committee (ACUC). For subcutaneous CT26tumor implantation, LPS and CLP experiments, female C57BL/6 and BALB/Cmice of 6-8 weeks were purchased from Harlan Laboratories. For anti-mCD3treatment, female BALB/C mice of 5-6 months old were purchased formHarlan laboratories. For the CART19 treatment, NSG-SGM3 (NSGS) mice(Stock no. 013062) were purchased from The Jackson Laboratory.

Chemicals and Reagents

For immunofluorescent staining, Alexa Fluor 594 goat anti-mouse and 488goat anti-rabbit IgG were purchased from Invitrogen. Anti-mCD3(145-2C11) and anti-Ly6G (8C5) antibodies were purchased from Bio XCell. α-methyl-D,L-p-tyrosine methyl ester hydrochloride (Santa CruzBiotechnology, SC-219470) is a soluble from of α-methyl-tyrosine(metyrosine) that is converted to α-methyl-tyrosine in vivo (see, e.g.,Corrodi et al., 1966 Psychopharmacologia, 10:116). LPS from Escherichiacoli 0111:B4 (L2630), (−)-epinephrine (E4250) and human ANP (A1663) werepurchased from Sigma.

Strain Engineering of C. novyi-NT

The site-specific knock-in of hANP in C. novyi-NT employed the TargeTronGene Knockout System (Sigma), which is based on the retrohomingmechanism of group II introns (see, e.g., Kuehne et al., 2012Bioengineered, 3:247). The sequence of the human ANP cDNA was optimizedfor Clostridium codon usage asTCATTAAGAAGATCTTCATGTTTTGGAGGAAGAATGGATAGAATAGGAGCTCAATCAGGATTAGGATGTAATTCATTCAGATATTAA (SEQ ID NO:2) coding for 28 AA(SLRRSSCFGGRMDRIGAQSGLGCNSFRY; SEQ ID NO:1). The synthesized sequencewas cloned into the shuttle vector pMTL8325. The construct included theC. novyi PLC signal peptide sequence under the control of the C. novyiflagellin promoter. Subsequently, the MluI fragment of the construct wassubcloned into the vector pAK001 (pMTL8325-pJ1R750aiReverse-pFla-153s-MCS-pThio-G1-ErmB) targeting the knock-in in the 153ssite of C. novyi-NT genome. The E. coli CA434 strain containing thetargeting construct was conjugated with C. novyi-NT and selected withpolymyxin B/erythromycin (Sigma) under anaerobic condition. Colonieswere selected and re-plated three times on non-selection plates andagain on the erythromycin plate. Clones were tested first by PCR usingEBS Universal and 153 S-F primers. Positive clones were further testedby PCR with primers targeting the backbone of the vector to confirm theinsert was integrated in C. novyi genome and with primers coveringexternally both sides of 153S to confirm the correct insertion. Thepropagation and sporulation of C. novyi-NT strains followed proceduresdescribed elsewhere (Bettegowda et al., 2006 Nature Biotechnology,24:1573-80).

ANP Measurement and cGMP Assay

ANP concentrations in the supernatant of ANP-C. novyi-NT culture and inmouse plasma were measured by an Elisa kit from Ray Biotech (EIAR-ANP-1)that recognizes both human and mouse ANP. ANP in the supernatant ofANP-C. novyi-NT culture were shown biological activities as describedelsewhere (Lofton et al., 1990 Biochem. Biophys. Res. Comm., 172:793-9).Briefly, bacterial supernatants were applied to cultured bovine aorticendothelial cells (BAOEC, Cell Applications Inc.) for 3 minutes. cGMPconcentrations were then measured in BAOEC lysates by the Direct cGMPElisa Kit from Enzo following the manufacture's instruction.

Subcutaneous Tumor Models and C. novyi Therapy

The colon cancer cell line CT26 was injected subcutaneously into theright flank of six to eight week old female Balb/C mice as describedelsewhere (Qiao et al., 2011 Oncotarget, 2:59-68). Tumor sizes weremeasured with a caliper and calculated as ½*L*W*H as described elsewhere(Tomayko et al., 1989 Can. Chemother. Pharmacol., 24:148-54). Whentumors reached 600-900 mm³ after about two weeks, 12×10⁶ spores of C.novyi-NT or ANP-C. novyi-NT at 3×10⁶/μl were injected intratumorallyinto 4 central parts of the tumor with a 32G Hamilton syringe needle.The bacteria typical germinated in the tumors within 24 hours, turningthem necrotic. Hydration of the mice was supported by daily subcutaneousinjections of 500 μl saline. Human ANP (Sigma) was dissolved in saline,loaded in mini-osmotic pumps (ALZET) with a release rate of 12 μg/dayand implanted subcutaneously in the back of mice 12 hours before thespore injection. Pumps loaded with saline served as controls. Metyrosinewas dissolved in PBS and injected IP at 60 mg/kg/day for three daysbefore the C. novyi injection to deplete catecholamines in storage. Twohours after the spore injection, 60 mg/kg of metyrosine was injectedintraperitoneally (IP). For each of the next three days, IP injectionsof metyrosine at 30 mg/kg were administered. Control groups wereinjected with PBS at the same time points.

Peritoneal Macrophage Experiments

Isolation of elicited macrophages from mouse peritoneum followedpreviously described procedures with minor modifications (Zhang et al.,“The isolation and characterization of murine macrophages,” Curr ProtocImmunol Chapter 14, Unit 14.1 (November 2008)). Four days prior to theharvest, 1 ml of 3% Brewer's thioglycollate medium (BD) was injected IPin female 2-3 months old BALB/c mice. Mice were euthanized by cervicaldislocation and the skin of the belly was cut open without penetratingthe muscle layer. Using a syringe with a 22G needle, 5 ml of cold PBScontaining 5 mM EDTA was injected carefully into the peritoneal cavity.After massaging gently for 1-2 minutes, a 1-ml syringe without needlewas used to extract the peritoneal contents containing residentialmacrophages. Cells were centrifuged at 400 g for 10 minutes at 4° C.,resuspended in DMEM/F12 medium supplemented with 1% FBS and antibioticsand distributed in 48-well plates at a concentration of 0.5×10⁶cells/well. After incubation at 37° C. for 2 hours, cells were rinsedthree times with 0.5 ml media and then 250 μl of media was added to eachwell. Ten minutes before the addition of LPS or epinephrine, metyrosineat 2 mM or ANP at 5 μg/ml was added to the cells. For stimulation, thecells were incubated for 24 hours with LPS at 50 μg/ml. An initialsolution of 3 mg/ml (−)-epinephrine was made with 0.1 N HCl andsubsequently diluted with PBS. To stimulate macrophages, they wereexposed to epinephrine at 15 ng/ml for 24 hours at 37° C. After theincubation, supernatants were collected from the wells and mixed with 5mM EDTA and 4 mM sodium metabisulfite for preservation of catecholaminesand stored at −80° C. Control experiments showed that all detectableepinephrine was degraded after incubation in media for 24 hours at 37°C. Thus, any epinephrine identified in the media must have been secretedby cells in the last 24 hours prior to harvesting the media.

LPS Experiments in Mice

LPS from Escherichia coli 0111:B4 was formulated as a 10 mg/ml solutionin water and stored in −80° C. LPS was injected intraperitoneally at adose of 3.5 mg/kg. This dose was found to be optimal for demonstratingthe protective effects of ANP and metyrosine. Human ANP (Sigma) wasdissolved in saline, loaded in mini-osmotic pumps (ALZET) with a releaserate of 12 μg/day and implanted subcutaneously in the back of mice 12hours before the LPS injection. Mice implanted with pumps loaded withsaline served as controls. Metyrosine was freshly dissolved in PBS andinjected IP at 60 mg/kg/day for three days prior to the LPS treatment.One hour before the LPS injection, metyrosine was injected at 60 mg/kginto the lower abdomen contralateral to the side of LPS injection. Thecontrol groups were injected with PBS. For the following 3 days,metyrosine was injected at 30 mg/kg/day IP. Hydration of mice wassupported by daily subcutaneous injection of 0.5 ml saline.

CLP Experiments

Cecal ligation and puncture (CLP) was performed as described elsewhere(Rittirsch et al., 2009 Nature Protocols, 4:31-6). Briefly, six-to-eightweek old female C57BL/6 mice were anesthetized and following abdominalincision, the cecum was ligated at about ¼ the distance from the luminalentry to its tip. The ligated cecum was punctured through and throughwith a 22G needle at ½ and ¾ the distance from the luminal entry to itstip. A small amount of the cecal content was gently pushed out of thefour openings into the peritoneum. Subsequently, the abdominal muscleswere sutured and the skin was closed with two staples. Five hundredmicroliters of saline were immediately injected subcutaneously to themice. For the groups treated with antibiotics, imipenem (Sigma) wasinjected subcutaneously at 25 mg/kg starting from 20 hours after CLP,with a schedule of twice a day on day one and once a day thereafter for10 days. Human ANP (Sigma) was dissolved in saline, loaded inmini-osmotic pumps (ALZET) with a release rate of 12 μg/day andimplanted subcutaneously in the back of mice 12 hours before the CLP,with pumps loaded with saline serving as controls. Metyrosine wasfreshly dissolved in PBS and injected IP at 60 mg/kg/day for three daysbefore the CLP. Twenty minutes before the CLP, metyrosine was injectedat 60 mg/kg IP into the right side. The control groups were injectedwith PBS. For the following 4 days, metyrosine was injected at 30mg/kg/day IP into the right side. Hydration of mice was supported bydaily subcutaneous injection of 0.5 ml saline.

Anti-CD3 Treatment

Five to six-month old Female BALB/c mice were used because we observedthat young mice treated with anti-CD3 antibodies underwent severe weightloss but did not consistently die, even at very high doses of theanti-CD3 antibody. Metyrosine was freshly dissolved in PBS and injectedIP at 60 mg/kg/day for three days prior to injection of anti-CD3antibodies. Various doses of anti-CD3 antibody were tested, and it wasfound that 125 μg/mouse resulted in the death of about half the mice;this was the dose chosen for further experiments. Thirty minutes beforethe IP injection of the anti-mouse CD3 antibody (BioXcell, 145-2C11),metyrosine was IP injected at 60 mg/kg into the contralateral side. Asingle additional dose of 30 mg/kg metyrosine was injected IP on thefollowing day. Control groups were injected with PBS at the same times.

In Vitro Assays of Raji and Anti-CD19 CAR-T Cells

Raji, a human Burkitt's lymphoma cell line, was purchased from Sigma.Human CD19scFv-CD28-4-1BB-CD3t CAR-T cells (PM-CAR1003) were purchasedfrom Promab Biotechnologies and maintained less than 7 days in AIM-Vmedium (GIBCO) supplemented with 300 IU/ml of hIL2 (Peprotech), 5% FBSand antibiotics (Car-T medium). In a 48 well plate, Raji cells wereplated at 1×10⁵/well and anti-CD19 CART cells were plated at 5×10⁵/wellin 275 μl of CAR-T medium. A solution of 3 mg/ml (−)-epinephrine wasmade in 0.1 N HCl and subsequently diluted in PBS for use at a finalconcentration of 15 ng/ml. Five minutes before the Raji and CART cellswith or without epinephrine were mixed, metyrosine at 2 mM or human ANPat 5 μg/ml was added and then the cells were incubated for 24 hours at37° C. After incubation, the cells were pelleted by centrifugation at700 g and 4° C. for 5 minutes and the supernatants were collected andmixed with 5 mM EDTA and 4 mM sodium metabisulfite for preservation ofcatecholamines, then stored at −80° C. until analysis.

Treatment of Tumor-Bearing Mice with Anti-CD19 CAR-T Cells

Six to eight-week old female NSG-SGM3 mice (NOD.Cg-Prkdcscid Il2rgtm1Wj1Tg(CMVIL3,CSF2,KITLG)1Eav/MloySzJ, Stock #013062) were purchased fromthe Jackson Laboratory. Raji cells were transfected with a luciferaseconstruct via lentivirus to create Raji-luc cells as described elsewhere(Bai et al., 2015 Neuro Oncol., 17:545). Human CD19scFv-CD28-4-1BB-CD3ζCART cells (PM-CAR1003, CART19) from Promab Biotechnologies weremaintained for less than 7 days in AIM-V medium (GIBCO) supplementedwith 300 IU/ml of hIL2 (Peprotech), 5% FBS and antibiotics. One daybefore the injection of Raji cells, mice were irradiated at a dose of 2Gy in a CIXD Xstahl device. One million Raji-luc cells were injected IVvia tail vein on day zero. Six days later, tumor loads were assessedusing a Xenogen instrument and 15×10⁶ CART19 cells were injected IV.Metyrosine was injected IP at 60 mg/kg/day for three days before theCART19 injection. On the day of CART19 injection, a fourth dose of 60mg/kg was given IP and the mice were subsequently injected four moretimes at daily intervals at 30 mg/kg.

Immunofluorescence and Immunohistochemistry Staining

Immunohistochemical (IHC) staining of paraffin-embedded mouse organs bythe rat anti-Ly6G (8C5) antibody was performed as described elsewhere(see, e.g., Bai et al., Neurooncology 2015, 17:545-54), with theexception that rabbit anti-rat IgG biotin (312-066-045, JacksonImmunoResearch) and Streptavidin peroxidase (Biogenex) were used assecondaries and staining reagents, respectively.

Measurement of Catecholamines and Cytokines in Mouse Plasma

Blood samples were collected into tubes containing 5 mM EDTA and 4 mMsodium metabisulfite after puncturing the facial vein or (terminally) bycardiac puncture. Subsequently, the samples were centrifuged and theplasmas were stored at −80° C. prior to analysis. Catecholamines(dopamine, norepinephrine and epinephrine) were measured using the 3-CATResearch ELISA kit from Labor Diagnostika Nord GmbH/Rocky MountainDiagnostics. Cytokines were measured using Luminex assays based onMillipore Mouse and Human Cytokine/Chemokine panels.

Bilateral Adrenalectomy of Mice

Adrenalectomy was performed with 6-8 week-old female BALB/c mice. Micewere anesthetized similarly to the procedure in CLP experiments and asmall incision was first made on one side of the back. After cuttingthrough the muscle and exposing the peritoneal cavity, adrenal gland wasidentified as a small and pink organ located near the anterior pole ofthe kidney. The whole adrenal gland was carefully removed by a scissorwith the help of forceps. The muscle was sutured and the skin was closedby a surgical stapler. Same procedure was repeated to the contralateraladrenal gland. Mice were given buprenorphine IP at 0.05 mg/kgimmediately and the following day for pain reduction and 0.5 ml salinesubcutaneously every day. Mice were allowed to recover for three daysbefore the next procedure.

Results

Experiments described herein employed the anaerobic spore-formingbacterial strain Clostridium novyi (C. novyi)-NT to treat cancer(Staedtke et al., Genes and Diseases 2016, 3:144-52). These bacteria arestrict anaerobes, and when spores are injected into animals or humans,bacteria germinate exclusively in hypoxic tumor tissues and can destroythem (Roberts et al., Science Transl. Med. 2014, 6:249ra111). However,when high doses of spores are injected into very large tumors, a massiveinfection occurs and the animals die within a few days from theconsequences of cytokine-related toxicity.

To mitigate dose-limiting toxicity, mice were pre-treated, prior toinjection of spores, with a variety of agents known to downregulate theinflammatory immune response, which has been highly effective in similarconditions (Grupp et al., New Engl. J. Med. 2013, 368:1509-18; Riedemannet al., J. Immunol. 2003, 170:503-7; Qiu et al., Critical Care Med.2013, 41:2419-29; Weber et al., Science 2015, 347:1260-5; Annane et al.,JAMA 2002, 288:862-71). Blocking antibodies to the receptors for thepro-inflammatory cytokines IL-6R or IL-3, and antibodies to circulatingTNF-α, had no effect on survival (FIG. 1). Similarly, theanti-inflammatory agent dexamethasone did not protect animals fromsepsis, even when used at very high doses.

The bacteria were engineered to remove bacterial components responsiblefor eliciting the overwhelming host immune response. All of thesestrains proved to germinate in tumors but none could eradicate tumorswhile sparing the mice.

The bacteria were then engineered to secrete atrial natriuretic peptide(ANP). To see if ANP could protect mice from massive bacterialinfections such as those caused by C. novyi-NT, C. novyi-NT wereengineered to express and secrete ANP. A gene cassette encoding the ANPof 28-amino acids (AA) fused with a signal peptide at the N-terminus wasoptimized for C. novyi codon usage. This gene cassette was stablyintegrated into the C. novyi-NT genome using a method that combined thegroup II Intron targeting and bacterial conjugation (see Methods).Selected C. novyi-NT clones were characterized for ANP expression (FIG.2A), biologic activity (FIG. 2B) and growth patterns in vitro (FIG. 2C).The clone with the highest expression of ANP, called ANP-C. novyi-NT,was selected for further studies.

A single dose of ANP-C. novyi-NT spores injected into subcutaneouslyimplanted CT26 colorectal tumors resulted in robust germination andcures, just as with the parental C. novyi-NT strain (FIG. 3A). PlasmaANP levels in mice injected with the ANP-C. novyi-NT strain wereincreased five times over that of mice injected with the parental C.novyi-NT strain (FIG. 2D). Strikingly, ˜80% of the animals receiving theANP-C. novyi-NT strain survived while none of the mice treated with theparental C. novyi-NT strain alone survived for longer than 5 days (FIG.3A, upper panel). Moreover, 87% of the surviving mice treated withANP-C. novyi-NT strain had complete tumor regressions and long-termcures (FIG. 3A lower panel).

There was a noticeable reduction of tissue damage and leukocyteinfiltration in the lungs, liver, and spleen of mice treated with ANP-C.novyi-NT (FIG. 1B). Likewise, mice injected with the ANP-C. novyi-NTstrain had significantly less inflammatory cytokines and chemokines intheir circulation than those treated with the parental strain. Inparticular, there were drastic reductions in cytokines and chemokinesreleased from activated T cells (IL-6, TNF-α) and monocytes/macrophages(IL-1β, IL-6, MIP-2, TNF-α), as well as chemoattractants (KC), cytokinesinvolved in tissue damage (IL-6, KC), and to a lesser degree in IFN-γ,MIP-1β, IL-10 and MCP-1 (FIG. 3C and FIG. 2E). Systemically deliveredANP also resulted in major reductions of pro-inflammatory cytokines inthe circulation, similar to what was observed with ANP-C. novyi-NT (FIG.3C and FIG. 2E).

This study was repeated in a different tumor type in another strain ofmice. Using subcutaneous implants of the glioblastoma cell line GL-261in C56B1/6 mice, 100% of mice treated with ANP-C. novyi-NT survived andhad substantial tumor reductions, while nearly all of the mice treatedwith the parental strain of C. novyi-NT died within 72 hours ofinfection (FIG. 4).

To determine whether protection from the CRS was due to the expressionof ANP rather than to some other unknown change in the engineeredstrain, mice were pre-treated with ANP released from an osmotic pumpthat was implanted 12 hours prior to injection with parental C. novyi-NTspores. The ANP delivered by this pump proved efficacious, with ˜75% ofthe mice surviving (FIG. 3A, upper panel). Of those that survived, allanimals showed significant therapeutic responses: in 80%, the tumor wascompletely eradicated and in the other 20%, a robust but not curativeresponse was observed (FIG. 3A, lower panel).

C. novyi-NT is a gram positive bacterium and it is known that sepsisresulting from gram-positive bacteria is different than that resultingfrom gram-negative bacteria with regards to host-immune interaction andcytokine release (Surbatovic et al., 2015 Sci. Rep., 5:11355).Genetically-engineered gram-negative bacteria are also being used inexperimental therapies for cancer (Zheng et al., 2017 Sci. Transl. Med.,9; Forbes, 2010 Nat. Rev. Cancer, 10:785-94; and Hoffman, 2016 MethodsMolecular Biology, 1409:177). To determine whether ANP could protectmice from infection with bacteria in general, its effects were evaluatedwhen administered prior to cecal ligation and puncture (CLP), aparticularly challenging sepsis model. This puncture releases largenumbers of enteric bacteria, including many species of gram-negativebacteria, into the peritoneum, causing polymicrobial peritoneal sepsis.ANP was administered as described above, using an osmotic pump that wasimplanted subcutaneously 12 hours before CLP. ANP significantly reducedthe mortality from the polymicrobial peritoneal sepsis—almost half ofthe animals survived the acute phase, while all animals died in thecontrol arm (FIG. 5A). Treatment with ANP after, rather than before, CLPdid not rescue the mice. Thus, ANP represents a method to preventtoxicity from cytokine release. In the ANP pre-treated mice, pathologicexamination revealed less peritoneal inflammation, pulmonary septalthickening, and hepatic inflammation compared to control animals (FIG.5B and FIG. 6A). The levels of pro-inflammatory cytokines present in thecirculation after ANP pretreatment was greatly reduced compared tocontrol animals following the CLP procedure. This reduction wasparticularly pronounced for cytokines IL-6, KC, MIP-2 and MIP-1β, andMCP-1 (FIG. 5C and FIG. 6B).

To investigate the mechanism underlying the protective effects of ANP,BMS-345541, a highly selective inhibitor of IκB kinase that has beenshown to reduce cytokine levels in other model systems (Burke et al.,2003 J. Biol. Chem., 278:1450), was administered to mice bearing largeCT26 tumors prior to intratumoral injection with the parental strain ofC. novyi-NT. However, unlike the case with ANP, there was no improvementin survival after pre-treatment with BMS-345541 (FIG. 7). This resultsuggested that ANP inhibits the hyperinflammation resulting from C.novyi-NT infection through means in addition to, or other than, thoseinvolving the NF-κB pathway.

To investigate a potential relationship between atecholamines and theprotective effects of ANP, it was first determined whether ANP couldinhibit the production of catecholamines in isolated macrophages. ANPwas found to reduce the production of all three major catecholamines(epinephrine, norepinephrine and dopamine) in mouse peritonealmacrophages exposed to inflammatory stimuli (FIG. 8A and FIG. 9A). Itwas also found that epinephrine itself can stimulate catecholamineproduction in macrophages in an autocrine manner and ANP pre-treatmentinhibited this production (FIG. 8C and FIG. 9A).

If the protective effects of ANP were due to its ability to interferewith catecholamine production, then inhibition of catecholaminesynthesis should mimic the effects of ANP. Pre-treatment withα-methyltyrosine (metyrosine), a specific inhibitor of catecholaminesynthesis, greatly reduced the catecholamines produced by mousemacrophages exposed to LPS, a potent inflammatory stimulus (FIG. 8A).Cytokine release by macrophages was similarly inhibited by metyrosine invitro (FIG. 8B). Next, mouse peritoneal macrophages were exposed tophysiologic levels of epinephrine or epinephrine plus LPS to demonstratethe inflammatory response of the autocrine induction of catecholaminesand cytokines, and suppression was similarly inhibited (FIGS. 8C and 8D;FIG. 10; FIG. 9A; and FIG. 11A).

Metyrosine was found to have similar effects in vivo. When mice werepre-treated with metyrosine and then administered the same inflammatorystimulant, ˜70% of the mice survived, whereas only 23% survived withoutmetyrosine pre-treatment (FIG. 10C). Both the levels of catecholaminesand the levels of inflammatory cytokines were substantially reduced inthe mice pre-treated with metyrosine (FIGS. 10D and 10E; FIG. 9B).

To document the generality of the effects of metyrosine, mice weretreated with metyrosine prior to the induction of CRS by infection withparental C. novyi-NT. 85% of the mice pre-treated with metyrosinesurvived while only 7% of the mice in the control arm survived (FIG.12A). As expected, catecholamines and cytokines were substantiallyreduced in animals pre-treated with metyrosine (FIGS. 12B and 12C; FIG.9C). Pre-treatment with metyrosine could also protect a subset (20%) ofmice from polymicrobial peritonitis (FIG. 12D), though less effectivelythan ANP (FIG. 5A). However, when mice were pre-treated with metyrosineas well as with the β-lactam antibiotic imipenem at 20 hours afterCLP, >⅔ of the mice survived CLP, while 88% of mice treated with theantibiotic alone died. This experiment highlights the fact that deathfrom overwhelming bacterial infections is due to two factors: thebacteria themselves and the host reaction to the infection (i.e. CRS).To formally demonstrate that the detrimental host response wasdiminished by metyrosine pre-treatment, the levels of circulatingcytokines were measured as described above. Multiple cytokinescharacteristic of sepsis or inflammation were substantially reduced bymetyrosine after infection with C. novyi-NT or induction ofpolymicrobial peritonitis by CLP (FIGS. 12C and 12F). The effects ofmetyrosine on circulating catecholamines were also documented (FIGS. 12Band 12E; FIGS. 9C and 9D).

CRS is also observed after the administration of therapeutics notinvolving bacteria. For example, immunotherapeutic agents targeting CD3molecules on the surface of T-cells is a promising treatment forautoimmune diseases and for the prevention of allograft rejection.However, the clinical implementation of such therapies (OKT3) has beenhampered by CRS resulting from generalized T-cell activation (Chatenoudet al., 1990 Transplantation, 49:697; and Guglielmi et al., 2016 ExpertOpin. Biolog. Ther., 16:841). To determine whether CRS unrelated tobacteria were accompanied by an increase in catecholamines,catecholamine levels were measured in mice at 24 and 48 hours afterinjection of an anti-CD3 antibody. The levels of epinephrine,norepinephrine and dopamine all increased substantially at both timepoints (FIG. 13A and FIG. 14A). Inflammatory cytokines also increasedsimilar to that observed after infection with C. novyi-NT or inductionof polymicrobial sepsis (FIGS. 12B and 12E). When mice were treated withmetyrosine prior to administering anti-CD3 antibodies, the levels ofcirculating catecholamines were reduced to near normal levels. Thisdecrease was associated with major reductions of the pro-inflammatorycytokines IL-6, TNF-α and KC among others (FIG. 13B and FIG. 14B). Mostimportantly, pre-treatment with metyrosine significantly impacted thesurvival of the mice: the majority of those treated only with anti-CD3antibodies died, while eleven of the twelve mice pre-treated withmetyrosine survived (FIG. 13C).

T-cell-mediated immunotherapies for cancer have recently been shown toachieve complete and durable tumor remissions in a subset of cancerpatients. B cell malignancies are the most common tumor types to beeffectively treated by such therapies; CD19-directed chimeric antigenreceptor-modified T-cells (CARTs) have generated response rates of up to95% in advanced cancers (Johnson et al., 2017 Cell Res., 27:38). Yet,the excessive and rapid tumor clearance as well as on-target, off-tumoractivation of the engineered T-cells have been associated withdose-limiting toxicities and occasionally even lethal CRS (Teachey etal., 2016 Can. Disc., 6:664-79; Fitzgerald et al., 2017 Crit. Care Med.,45:e124-e31; Grupp et al., 2013 New Eng. J. Med., 368:1509-18; Lee etal., 2014 Blood, 124:188-95; and Maude et al., 2014 New Eng. J. Med.,371:1507-17). To investigate whether CD19-directed CART (CART19) cangenerate and release significant catecholamines during tumor cellkilling, the Burkitt's lymphoma-derived Raji cells were incubated withCART19. Levels of epinephrine and norepinephrine as well as variouscytokines in culture supernatants increased substantially at 24 hoursafter exposure to CART19 (FIGS. 13D and 13E). The surge incatecholamines and cytokines was even more impressive when exogenousepinephrine was added to the cells (FIGS. 11A and 11B). Blockade ofcatecholamine synthesis with ANP and metyrosine significantly decreasedthe production of catecholamines and subsequent inflammatory responsesas defined by cytokine production (FIGS. 13D and 13E; FIGS. 11A and11B). To investigate the effect of catecholamine suppression on CRS invivo, human Raji cells engrafted in the NSG™-SGM3 (NSGS) mice wereallowed to grow for 6 days to establish a significant tumor burdenbefore treatment with CART19. Blood obtained at 24 and 72 hours revealedpeak levels of catecholamines and systemic release of various human andmouse cytokines, including IL-6, IFN-γ, TNF-α, KC, and MIP-2, which weresignificantly reduced when the mice had been pre-treated with metyrosine(FIGS. 13F and 13G; FIG. 11C).

A model explaining the reduced biotherapeutic toxicity resulting frompre-treatment with metyrosine is depicted in FIG. 15. Briefly, the datadescribed herein suggest that catecholamines drive CRS via aself-amplifying feed-forward loop in immune cells such as macrophagesand T-cells. Catecholamines secreted by immune cells andcatecholamine-producing organs bind to the adrenergic receptors on theimmune cells, stimulating more catecholamine and cytokine release,recruiting other inflammatory cells to the sites of inflammation, andeventually leading to organ system failure and death. Most importantly,this self-amplifying feed-forward loop has a central node—tyrosinehydroxylase—which, as demonstrated herein, can be exploited to interruptthe feed-forward loop, thereby modulating the inflammatory response.

Example 2 Reducing Mortality from Therapy-Induced Cytokine 1 ReleaseSyndrome via Disruption of a Self-Amplifying Catecholamine SynthesisLoop Materials and Methods Mice

All animal works were performed in accordance with protocols specifiedby the Johns Hopkins Animal Care and Use Committee (ACUC). Forsubcutaneous CT26 tumor implantation, LPS and CLP experiments, femaleC57BL/6 and BALB/C mice of 6-8 weeks were purchased from HarlanLaboratories. For anti-mCD3 treatment, female BALB/C mice of 5-6 monthsold were purchased form Harlan laboratories. For the CART19 treatment,NSG-SGM3 (NSGS) mice (Stock no. 013062) were purchased from the JacksonLaboratory.

LysMcre-Conditional TH Knockout Mice

LysMcre mice were purchased from Jackson 596 Laboratory (stock no.004781), in which a nuclear-localized Cre recombinase was inserted intothe first coding exon of the lysozyme 2 gene and expressed in themyeloid cell lineage (monocytes, mature macrophages and granulocytes).TH loxP/loxP (TH fl/fl) mice were as described elsewhere (see, e.g.,Jackson et al., 2012 J Neurosci 32:9359-9368). By crossing these twostrains, LysMcre: TH fl/fl mice (TH^(ΔLysM)) were produced asexperimental strain for LPS and anti-CD3 experiments and LysMcre: TH+/+mice (TH+/+) were used as the Cre transgene control.

Chemicals and Reagents

Anti-mCD3 (145-2C11), anti-Ly6G (8C5) and anti-mIL6 receptor (15A7)antibodies were purchased from BioXcell. Anti-mTNFα antibody (R023) waspurchased from Sino Biological and anti-mIL3 antibody (MP2-8F8) waspurchased from BD Biosciences. α-methyl-D,L-p-tyrosine methyl esterhydrochloride (Santa Cruz Biotechnology, SC-219470) is a soluble from ofα-methyl-tyrosine (metyrosine) that is converted to α-methyl-tyrosine invivo, whereas the less soluble α-methyl-tyrosine was purchased fromSigma (120693). LPS from Escherichia coli 0111:B4 (L2630),(−)-epinephrine (E4250), dopamine (H8502), norepinephrine (A7256),prazosin (P7791), metoprolol (M5391) and human ANP (A1663) werepurchased from Sigma. RX 821002 (1324) and ICI 118551 (0821) werepurchased from Tocris.

Strain Engineering of C. novyi NT

The site-specific knock-in of human ANP in C. novyi-NT employed theTargeTron Gene Knockout System (Sigma), which is based on theretrohoming mechanism of group II introns. The sequence of the human ANPcDNA was optimized for Clostridium codon usage asTCATTAAGAAGATCTTCATGTTTTGGAGGAAGAATGGATAGAATAGGAGCTCAATCAGGATTAGGATGTAATTCATTCAGATATTAA (SEQ ID NO:2) coding for 28 AA(SLRRSSCFGGRMDRIGAQSGLGCNSFRY; SEQ ID NO:1). The synthesized sequencewas cloned into the shuttle vector pMTL8325. The construct included theC. novyi PLC signal peptide sequence under the control of the C. novyiflagellin promoter. Subsequently, the MluI fragment of the construct wassubcloned into the vector pAK001 (pMTL8325-pJIR750aiReverse-pFla-153s-MCS-pThio-G1-ErmB) targeting the knock-in in the 153ssite of C. novyi-NT genome. The E. coli CA434 strain containing thetargeting construct was conjugated with C. novyi-NT and selected withpolymyxin B/erythromycin (Sigma) under anaerobic condition. Colonieswere selected and re-plated three times on non-selection plates andagain on the erythromycin plate. Clones were tested first by PCR usingEBS Universal and 153 S-F primers. Positive clones were further testedby PCR with primers targeting the backbone of the vector to confirm theinsert was integrated in C. novyi genome and with primers coveringexternally both sides of 153S to confirm the correct insertion. Thepropagation and sporulation of C. novyi-NT strains followed proceduresdescribed elsewhere (see, e.g., Bettegowda et al. 2006 Nat Biotechnol24:1573-1580).

RNA Extraction and Quantitative PCR of C. novyi-NT Strains

RNA of germinated C. novyi-NT strains were extracted using RiboPureBacterial RNA Purification Kit (Ambion) and transcribed with SuperScriptIV RT Kit (Invitrogen) as described elsewhere (see, e.g., Bettegowda etal. 2006 Nat Biotechnol 24:1573-1580). Real-time PCR was performed usingMaxima SYBR Green/ROX qPCR Master Mix (Thermo Fisher), targeting on theNT01CX1854 gene specific for geminating C. novyi-NT (see, e.g.,Bettegowda et al. 2006 Nat Biotechnol 24:1573-1580).

ANP Measurement and cGMP Assay

ANP concentrations in the supernatant of ANP-C. novyi-642 NT culture andin mouse plasma were measured with an Elisa kit from Ray Biotech(EIAR-ANP-1) that recognizes both human and mouse ANP. ANP in thesupernatant of ANP-C. novyi-NT culture were shown to have biologicalactivity (see, e.g., Lofton et al., 1990 Biochem Biophys Res Commun172:793-799). Briefly, bacterial supernatants were applied to culturedbovine aortic endothelial cells (BAOEC, Cell Applications Inc.) for 3minutes. cGMP concentrations were then measured in BAOEC lysates by theDirect cGMP Elisa Kit from Enzo following the manufacture's instruction.

Subcutaneous Tumor Models and C. novyi-NT Therapy

The colon cancer cell line CT26 was injected subcutaneously into theright flank of six to eight weeks old female Balb/C mice as describedelsewhere (see, e.g., Qiao et al. 2011 Oncotarget 2:59-68). Tumor sizeswere measured with a caliper and calculated as ½ *L*W*H as describedelsewhere (see, e.g., Tomayko et al. 1989 Cancer Chemother Pharmacol24:148-154). When tumors reached 600-900 mm³ after about two weeks,12×10⁶ spores of C. novyi-NT or ANP-C. novyi-NT at 3×10⁶/μl wereinjected intratumorally into 4 central parts of the tumor with a 32GHamilton syringe needle. The bacteria typical germinated in the tumorswithin 24 hours, turning them necrotic. Hydration of the mice wassupported by daily subcutaneous injections of 500 μl saline. Human ANP(Sigma) was dissolved in saline, loaded in mini-osmotic pumps (ALZET)with a release rate of 12 μg/day and implanted subcutaneously in theback of mice 12 hours before the spore injection. Pumps loaded withsaline served as controls. Metyrosine was dissolved in PBS and injectedIP at 60 mg/kg/day for three days before the C. novyi injection todeplete catecholamines in storage. Two hours after the spore injection,60 mg/kg of metyrosine was injected intraperitoneally (IP). For each ofthe next three days, IP injections of metyrosine at 30 mg/kg wereadministered. Control groups were injected with PBS at the same timepoints.

Immunohistochemistry

Immunostaining for CD11b was performed on formalin-fixed, paraffinembedded sections on a Ventana Discovery Ultra autostainer (RocheDiagnostics) by Ms. Sujayita Roy of JHU Oncology Tissue Services.Briefly, following dewaxing and rehydration on board, epitope retrievalwas performed using Ventana Ultra CC1 buffer (#6414575001, RocheDiagnostics) at 96° C. for 64 minutes. Primary antibody, anti-CD11b(1:8000 dilution; catalog# ab133357, Abcam) was applied at 36° C. for 40minutes. Primary antibodies were detected using an anti-rabbit HQdetection system (#7017936001 and 7017812001, Roche Diagnostics)followed by Chromomap DAB IHC detection kit (#5266645001, RocheDiagnostics), counterstaining with Mayer's hematoxylin, rehydration andmounting.

In Vitro Macrophage Experiments

Isolation of elicited macrophages from mouse peritoneum followedpreviously described procedures with minor modifications66. Four daysprior to the harvest, 1 ml of 3% Brewer's thioglycollate medium (BD) wasinjected IP in female 2-3 months old BALB/c mice or 4-6 weeks oldconditional TH knockout mice. Mice were euthanized by cervicaldislocation and the skin of the belly was cut open without penetratingthe muscle layer. Using a syringe with a 25G needle, 5 ml of cold PBScontaining 5 mM EDTA was injected carefully into the peritoneal cavity.After massaging gently for 1-2 minutes, a 1-ml syringe without needlewas used to extract the peritoneal contents containing residentialmacrophages. Cells were centrifuged at 400 g for 10 minutes at 4° C.,resuspended in DMEM/F12 medium supplemented with 1% FBS and antibioticsand distributed in 48-well plates at a concentration of 0.5×10⁶cells/well. After incubation at 37° C. for 2 hours, cells were rinsedthree times with 0.5 ml media and then 250 μl of media was added to eachwell. Ten minutes before the addition of LPS 688 or epinephrine,metyrosine at 2 mM or ANP at 5 μg/ml was added to the cells. Forstimulation, the cells were incubated for 24 hours with LPS at 50 μg/ml.An initial solution of 3 mg/ml (−)-epinephrine was made with 0.1 N HCland subsequently diluted with PBS. To stimulate macrophages, they wereexposed to epinephrine at 15 ng/ml for 24 hours at 37° C. After theincubation, supernatants were collected from the wells and mixed with 5mM EDTA and 4 mM sodium metabisulfite for preservation of catecholaminesand stored at −80° C. Control experiments showed that all detectableepinephrine was degraded after incubation in media for 24 hours at 37°C. Thus, any epinephrine identified in the media must have been secretedby cells in the last 24 hours prior to harvesting the media.

Human U937 cells were cultured in RPMI 1640 media with 5% FBS andantibiotics, and were differentiated to M1 macrophage-like cells byincubating with 20 nM phorbol 12-myristate 13-acetate (PMA, Sigma) for24 hours and further culturing in RPMI 1640 media with 5% FBS andantibiotics for another 72 hours. The experiments with U937 were set upin the same way as described above with peritoneal macrophages. Tenminutes before the addition of LPS or epinephrine, metyrosine at 2 mM orANP at 5 μg/ml was added to the cells. Cells were incubated for 24 hourswith LPS at 1 μg/ml.

LPS Experiments in Mice

LPS from Escherichia coli 0111:B4 was formulated as a 10 mg/ml solutionin water and stored in −80° C. In Balb/C mice, LPS was injectedintraperitoneally at a lethal dose of 3.5 mg/kg. This lethal dose wasfound to cause 70-90% death rate and be optimal for demonstrating theprotective effects of ANP and metyrosine. In experiments withcatecholamine pumps, a sublethal dose with 15-35% death rate wasoptimized in Balb/C mice. In TH+/+ and TH^(ΔLysM) mice with C57BL/6background, a lethal dose was optimized at 5 mg/kg. Human ANP (Sigma)was dissolved in saline, loaded in mini-osmotic pumps (ALZET) with arelease rate of 12 μg/day and implanted subcutaneously in the back ofmice 12 hours before the LPS injection. Mice implanted with pumps loadedwith saline served as controls. Metyrosine was freshly dissolved in PBSand injected IP at the indicated doses for three days prior to the LPStreatment. One hour before the LPS injection, metyrosine was injectedinto the lower abdomen contralateral to the side of LPS injection. Thecontrol groups were injected with PBS. For the following 3 days,metyrosine was injected IP at reduced indicated doses. Hydration of micewas supported by daily subcutaneous injection of 0.5 ml saline.

CLP Experiments

CLP was performed as described described elsewhere (see, e.g., Rittirschet al., 2008 Rev Immunol 8:776-787). Briefly, 6-8 weeks old femaleC57BL/6 mice were anesthetized and following abdominal incision, thececum was ligated at about ¼ the distance from the luminal entry to itstip. The ligated cecum was punctured through and through with a 22Gneedle at ½ and ¾ the distance from the luminal entry to its tip. Asmall amount of the cecal content was gently pushed out of the fouropenings into the peritoneum. Subsequently, the abdominal muscles weresutured and the skin was closed with two staples. Five hundredmicroliters of saline were immediately injected subcutaneously to themice. For the groups treated with antibiotics, imipenem (Sigma) wasinjected subcutaneously at 25 mg/kg starting from 20 hours after CLP,with a schedule of twice a day on day one and once a day thereafter for10 days. Metyrosine was freshly dissolved in PBS and injected IP at60mg/kg/day for three days before the CLP. Twenty minutes before theCLP, metyrosine was injected at 60 mg/kg IP into the right side. Thecontrol groups were injected with PBS. For the following 4 days,metyrosine was injected at 30 mg/kg/day IP into the right side.Hydration of mice was supported by daily subcutaneous injection of 0.5ml saline.

Anti-CD3 Treatment

For survival experiments, five to six-month old Female BALB/c mice wereused because we observed that young mice treated with anti-CD3antibodies underwent severe weight loss but did not consistently die,even at very high doses of the anti-CD3 antibody. Metyrosine was freshlydissolved in PBS and injected IP at 60 mg/kg/day for three days prior toinjection of anti-CD3 antibodies. Various doses of anti-CD3 antibodywere tested, and it was found that 125 μg/mouse resulted in the death ofabout half the mice; this was the dose chosen for further experiments.Thirty minutes before the IP injection of the anti-mouse CD3 antibody(BioXcell, 145-2C11), metyrosine was IP injected at 60 mg/kg into thecontralateral side. A single additional dose of 30 mg/kg metyrosine wasinjected IP on the following day. Control groups were injected with PBSat the same times. For experiments with conditional TH knockout mice,4-6 week-old LysMcre: TH fl/fl (TH^(ΔLysM)) mice with C57BL/6 backgroundwere used and LysMcre: TH+/+ mice of the same age were used as control.In these experiments, 200 μg/mouse anti-mouse CD3 antibody was injectedIP.

Human Anti-CD19 CART (hCART19) Cells and Untransduced T Cells

Human CD19scFv-CD28-4-1BB-CD3ζ CAR-T cells (PM-CAR1003) were purchasedfrom Promab Biotechnologies and stored in liquid nitrogen upon delivery.The CAR construct includes a scFv derived fromFMC63 anti-CD19 antibody,a hinge region and a transmembrane domain of CD28 in a third-generationCAR cassette. Generation of CAR-encoding lentivirus, isolation,expansion and transduction of human T cells followed proceduresdescribed elsewhere (see, e.g., Berahovich et al., 2017 Front Biosci22:1644-1654). Cells were proliferated for two weeks in mediumcontaining 300 IU/ml of hIL2. CART cells were used freshly upondefrosting or maintained less than 7 days in the CART medium consistingof AIM-V medium (GIBCO) supplemented with 5% FBS (Sigma) andpenicillin-streptomycin (GIBCO), with the addition of 300 IU/ml of hIL2(Peprotech).

Untransduced T cells were purchased from ASTARTE Biologics(#1017-3708OC17, CD3+) and were used freshly upon defrosting ormaintained less than 7 days in CART medium.

In Vitro Assays of hCART19 Cells

Raji, a human Burkitt's lymphoma cell line, was purchased from Sigma. Ina 48 well plate, Raji cells were plated at 1×10⁵/well and hCART19 cellsor untransduced T cells were plated at 5×10⁵/well in 275 μl of medium. Asolution of 3 mg/ml (−)-epinephrine was made in 0.1 N HCl andsubsequently diluted in PBS for use at a final concentration of 15ng/ml. Five minutes before the Raji and CART cells with or withoutepinephrine were mixed, metyrosine at 2 mM or human ANP at 5 μg/ml wasadded and then the cells were incubated for 24 hours at 37° C. Controlexperiments showed that all detectable epinephrine was degraded afterincubation in media for 24 hours at 37° C. Thus, any epinephrineidentified in the media must have been secreted by cells in the last 24hours prior to harvesting the media. Cycloheximide (CHX, Sigma) wasadded at 10 μg/ml to Raji and CART cells 30 minutes before they weremixed. After incubation, the cells were pelleted by centrifugation at700 g and 4° C. for 5 minutes and the supernatants were collected andmixed with 5 mM EDTA and 4 mM sodium metabisulfite for preservation ofcatecholamines, then stored at −80° C. until analysis.

Treatment of Raji Tumor-Bearing Mice with hCART19 Cells

Six to eight weeks old female NSG-SGM3 (NSGS) mice (NOD.Cg-PrkdcscidIl2rgtm1Wj1Tg (CMV-IL3, CSF2, KITLG) 1Eav/MloySzJ, Stock #013062) werepurchased from the Jackson Laboratory. Raji cells were transfected witha luciferase construct via lentivirus to create Raji-luc cells. NSGS isa triple transgenic strain expressing human IL3, GM-CSF and SCF combinethe features of the highly immunodeficient NOD scid gamma (NSG) mouse.One day before the injection of Raji cells, mice were irradiated at adose of 2 Gy in a CIXD Xstahl device. In high tumor burden experimentsin FIG. 5, 10⁶ Raji-luc cells were injected IV via tail vein. Six dayslater, tumor loads were assessed using a Xenogen instrument and 15×10⁶hCART19 cells or untransduced T-cells were injected IV. In low tumorburden experiments in FIG. 6, 2×10⁵ Raji-luc cells were injected IV viatail vein. Four days later, tumor loads were assessed using a Xenogeninstrument and 15×10⁶ hCART19 cells were injected IV. Metyrosine wasinjected IP at 60 mg/kg/day for three days before the hCART19 injection.On the day of CART19 injection, a fourth dose of 60 mg/kg was given IPand the mice were subsequently injected four more times at dailyintervals at 30 mg/kg.

Mouse Anti-CD19 CART Cells (mCART19) and Untransduced T Cells

Mouse CD19scFv-CD28-CD3ζ CAR (m1928z) construct with GFP in SFGretroviral vector was as described elsewhere (see, e.g., Davila et al.,2013 PLoS One 8:e61338). The isolation, activation, and transduction ofmouse T cells followed the procedure described elsewhere (see, e.g.,Davila et al., 2013 PLoS One 8:e61338; and Lee et al., 2009 Methods MolBiol 506:83-96). Briefly, the spleens were harvested from female C57BL/6mice and T cells were enriched from splenocytes by passage over a nylonwool column (Polysciences, Warrington, Pa.). Mouse T cells were thenactivated with CD3/803 CD28 Dynabeads (Thermo Fisher) following themanufacturer's instructions and cultured in the presence of hIL2 at 30IU/mL (R & D Systems). Retrovirus was produced by transfectingPhoenix-Eco packaging cells (ATCC) and spinoculations were done twicewith retroviral supernatant. mCART19 cells were expanded for 10-14 daysas described elsewhere (see, e.g., Lee et al., 2009 Methods Mol Biol506:83-96). Untransduced T cells were produced following the sameprocedure without viral transduction.

Treating B Cell Acute Lymphoblastic Leukemia (B-ALL) with mCART19 inImmunocompetent Mice

The Eμ-ALL cell line was derived from a lymphoid malignancy in an Eμ-myctransgenic mouse and upon IV injection, can develop B-ALL in C57BL/6mice. The Eμ-ALL cells were co-cultured with feeder NIH-3T3 cells thatwere irradiated at 60 Gy, in RPMI 1640 media supplemented with 10% FBS,0.05 mM 2-Mercaptoethanol and antibiotics. Eμ-ALL cells were transfectedwith luciferase via lentivirus. 2×10⁶Eμ-ALL cells were IV injected infemale 6-8 week-old C57BL/6 mice via tail vein and after 6 days, micewere IP injected with cyclophosphamide (CPA) at 100 mg/kg forpre-conditioning as described elsewhere (see, e.g., Davila et al., 2013PLoS One 8:e61338). One day after CPA treatment, 10×10⁶ mCART19 cellswere IV injected in the mice. Metyrosine was injected IP at 40 mg/kg/dayfor three days before the mCART19 injection. On the day of mCART19injection, a fourth dose of 40 mg/kg was given IP and the mice weresubsequently injected four more times at daily intervals at 30 mg/kg.One day before mCART19 injection, mini-osmotic pumps (ALZET) loaded withhANP with a release rate of 12 μg/day were implanted subcutaneously inthe back of mice. Tumor load was monitored by Xenogen before and aftermCART19 injection.

Measurement of Catecholamines and Cytokines in Mouse Plasma

Blood samples were collected into tubes containing 5 mM EDTA and 4 mMsodium metabisulfite after puncturing the facial vein or (terminally) bycardiac puncture. Subsequently, the samples were centrifuged and theplasmas were stored at −80° C. prior to analysis. Catecholamines(dopamine, norepinephrine and epinephrine) were measured using the 3-CATResearch ELISA kit from Labor Diagnostika Nord GmbH/ Rocky MountainDiagnostics. Cytokines were measured using Luminex assays based onMillipore Mouse and Human Cytokine/Chemokine panels or ELISA kits formouse or human IL-6, TNF-α, MIP-1α, KC and IL-2 (R&D Systems) permanufacturer's instructions.

Results

The study reported here began with experiments employing the anaerobicspore-forming bacterial strain C. novyi-NT to treat cancer (see, e.g.,Staedtke et al., 2016 Genes and Diseases 3:144-152). These bacteria arestrict anaerobes, and when spores are injected into animals or humans,bacteria germinate exclusively in hypoxic tumor tissues and can destroythem (see, e.g., Roberts et al. 2014 Sci Transl Med 6:249ra111).However, when very high doses of spores are injected into very largetumors, a massive infection occurs and the animals die within a few dayswith severe cytokine release due to a combination of tumor lysis anddirect toxic effects of the bacteria (sepsis; see, e.g., Agrawal et al.,2004 Proc Natl Acad Sci USA 101:15172-15177; and Diaz Jr. et al., 2005Toxicol Sci 88:562-575). To mitigate this dose-limiting toxicity,pre-treating mice, prior to injection of spores, with a variety ofagents known to downregulate the inflammatory immune response wasattempted (see, e.g., Grupp et al., 2013 N Engl J Med 368:1509-1518;Riedemann et al., 2003 J Immunol 170:503-507; Qiu et al., 2013 Crit CareMed 41:2419-2429; Weber et al., 2015 Science 347:1260-1265; and Annaneet al., 2002 JAMA 288:862-871). Unfortunately, blocking antibodies tothe receptors for the pro-inflammatory cytokines IL-6R or IL-3, andantibodies directed against circulating TNF-α, had limited effects onsurvival with only anti-IL-6R showing a significant but marginalimprovement (FIG. 16A). Similarly, the antibiotic metronidazole andanti-inflammatory agent dexamethasone did not protect animals fromsepsis, even when used at very high doses.

Engineering the bacteria to secrete various anti-inflammatory proteinsthat might mitigate the bacteria-associated toxicity was then attempted;atrial natriuretic peptide (ANP) was the only protein that provedsuccessful in these experiments without compromising the efficacy. Tosee if ANP could protect mice from massive bacterial infections such asthose caused by C. novyi-NT, C. novyi-NT was engineered to express andsecrete ANP. A gene cassette encoding the ANP of 28-AA fused with asignal peptide at the N-terminus was optimized for C. novyi codon usage.This gene cassette was stably integrated into the C. novyi-NT genomeusing a method that combined the group II Intron targeting and bacterialconjugation (see Methods). Selected C. novyi-NT clones werecharacterized for ANP expression, biologic activity, and growth patternsin vitro (FIGS. 16B-16D). The clone with the highest expression of ANP,called ANP-C. novyi-NT, was selected for further studies.

A single dose of ANP-C. novyi-NT spores injected into subcutaneouslyimplanted CT26 colorectal tumors resulted in robust germination andcures. Levels of both plasma ANP and cGMP in mice injected with theANP-C. novyi-NT strain were increased two to four times over that ofmice injected with the parental C. novyi-NT strain (FIGS. 16E and 16F).Strikingly, at similar efficiencies of germination and proliferationbetween the two strains (FIG. 16G), ˜80% of the animals receiving theANP-C. novyi-NT strain survived while none of the mice treated with theparental C. novyi-NT strain alone did (FIG. 17A). Moreover, 84% of thesurviving mice treated with ANP-C. novyi-NT strain had complete tumorregressions and long-term cures (FIG. 17A).

There was a noticeable reduction of tissue damage and inflammatoryeffects in the liver, spleen, and lungs of ANP-C. novyi-NT treated mice,as demonstrated by fewer infiltrating CD11b positive (CD11b+) myeloidcells in these organs and a less elevated pulmonary permeabilitycompared to control animals that were given C. novyi-NT (FIGS. 16H-17B).Those mice had evidence of severe inflammatory changes, manyinfiltrating CD11b+ myeloid cells in the liver and lungs correlatingwith a high pulmonary permeability index (FIGS. 16H-17B).

Likewise, mice injected with the ANP-C. novyi-NT strain hadsignificantly less inflammatory cytokines and chemokines in theircirculation than those treated with the parental strain. In particular,there were drastic reductions in cytokines and chemokines released fromactivated myeloid cells (IL-1β, IL-6, MIP-2, TNF-α), as well aschemoattractants (KC), and to a lesser degree in IFN-γ, MIP-1β, IL-12,IL-10 and MCP-1 (FIG. 17C). Interestingly, the cytokine reductionsobserved in ANP-C. novyi-NT treated mice were accompanied bysignificantly lower levels of circulating catecholamine compared to micetreated with C. novyi-NT (FIGS. 16K and 17D). This finding did notappear to be related to changes in the volume homeostasis as estimatedplasma and volume hematocrit were similar in animals treated with C.novyi-NT and ANP-C. novyi-NT at 36 hours (FIGS. 16L and 16M). We nextrepeated the study in a different tumor type in another strain of mice.Using subcutaneous implants of the glioblastoma cell line GL-261 inC56B1/6 mice, we found that 100% of mice treated with ANP-C. novyi-NTsurvived and had substantial tumor reductions, while nearly all of themice treated with the parental strain of C. novyi-NT died within 72hours of infection (FIG. 18A).

It was then sought to determine whether the protective effect was due tothe expression of ANP rather than to some other unknown change in theengineered strain. For this purpose, mice were pre-treated with ANPreleased from an osmotic pump that was implanted 12 hours prior toinjection with parental C. novyi-NT spores. The ANP delivered by thispump proved efficacious, with ˜73% of the mice surviving, even though itdid not have any effect on tumor colonization of C. novyi-NT (FIG. 17A).Of those that survived, all animals showed significant therapeuticresponses: in ˜80%, the tumor was completely eradicated and in the other23%, a robust but not curative response was observed (FIG. 17A).Systemically delivered ANP also resulted in major reductions ofcirculating pro-inflammatory cytokines and catecholamines as well astissue injury, similar to what was observed with ANP-C. novyi-NT (FIGS.16J and 17C).

It was then sought to investigate the mechanism underlying theprotective effects of ANP. BMS-345541 was administered to mice bearinglarge CT26 tumors prior to intra-tumoral injection with the parentalstrain of C. novyi-NT. However, unlike the case with ANP, there was nosignificant improvement in survival after pre-treatment with BMS-345541(FIG. 18B). This result suggested that ANP inhibits thehyperinflammation resulting from C. novyi-NT infection through means inaddition to, or other than, those involving the NF-κB pathway. In thisregard, the changes to the catecholamine levels during the bacterialtherapy were particularly intriguing and prompted us to investigatetheir relationship to the protective effects of ANP.

It was determined which catecholamines contributed to the CRS severity.For this purpose, subcutaneously implanted osmotic pumps thatcontinuously released epinephrine, norepinephrine or dopamine into thecirculation of mice treated with the potent inflammatory stimulant LPSwere used. Mice co-treated with epinephrine had an earlier onset andexacerbated disease course, as demonstrated by increased mortality andelevated levels of catecholamines and IL-6, TNF-α, MIP-2, and KC,compared with that of LPS-only treated mice (FIGS. 19A-19D). This effectwas less pronounced in animals co-treated with norepinephrine and absentwith dopamine. Furthermore, it was found that epinephrine can moderatelystimulate cytokine release of IL6, KC and MIP-2, even though no effecton animal survival was observed (FIGS. 19A-19D).

Next, it was investigated the catecholamine synthesis in mouseperitoneal macrophages. ANP was found to suppress the increasedproduction of the macrophageal catecholamines (epinephrine,norepinephrine and dopamine) induced by LPS and this suppressioncorrelated with a reduction in the cytokine levels of IL-6, TNF-α, 162MIP-2, and KC compared to the controls (FIGS. 20A-20B). Peritonealmacrophages treated with epinephrine alone also showed a mild butnoticeable upregulation of the catecholamine and cytokine (IL-6, TNF-α,MIP-2, and KC) production, which was also suppressed by ANP (FIGS.19F-19G). Subsequently, epinephrine in combination with LPS vigorouslyaugmented the inflammatory response and this amplification was inhibitedby ANP (FIGS. 20A-20B).

If the protective effects of ANP were due to its ability to interferewith catecholamine production, then inhibition of catecholaminesynthesis should mimic the effects of ANP. α-methyltyrosine (metyrosine)is a specific inhibitor of catecholamine synthesis. Metyrosine binds tothe active site of tyrosine hydroxylase (TH) and prevents binding of itsnatural substrate L-tyrosine, thereby inhibiting its conversion toL-dihydroxyphenylalanine (L-DOPA), the precursor of dopamine,norepinephrine and epinephrine. Pre-treatment with metyrosine greatlyreduced the catecholamines produced by mouse macrophages exposed to LPS,epinephrine or the combination of both (FIGS. 19F-19G; FIGS. 20A-20B).Consistent with this, the cytokines released by macrophages werediminished by metyrosine pretreatment (FIGS. 19F-19G; FIGS. 20A-20B).Comparable results were obtained using human U937-derived macrophagecells (FIG. 21A).

To further confirm that the production of catecholamines frommacrophages drives the inflammatory response, isolated peritonealmacrophages from mice with selective deletion of the gene encoding TH inLysM+ myeloid cells (LysMcre:TH fl/fl or THD^(ΔLysM)) were used. Thesemice showed significantly reduced TH expression in the harvestedperitoneal macrophages (FIG. 21C) but not in lymphoid cell populations.Peritoneal macrophages with myeloid-specific deletion of TH showed areduced secretion of catecholamines and inflammatory cytokines uponstimulation with LPS and epinephrine, which confirmed the role forautocrine catecholamine production in macrophages in the amplificationof the inflammatory cascade (FIGS. 21C-21E). Consistently, theLPS-induced production of catecholamines as well as inflammatorycytokines was abrogated in the THD^(ΔLysM) mice that showed a survivalbenefit of 66%, while >65% of control mice died from the lethal toxicity(FIGS. 20C-20E).

Metyrosine was found to have similar effects in vivo. When mice werepre-treated with metyrosine and then administered the same inflammatorystimulant, ˜75% of the mice survived, whereas only 10% survived withoutmetyrosine pre-treatment (FIG. 22). Both the levels of catecholaminesand the levels of inflammatory cytokines were substantially reduced inthe mice pre-treated with metyrosine (FIG. 22). The efficacy ofmetyrosine was dose-dependent; animal survival correlated with thedegree of catecholamine depletion and cytokine reductions (FIG. 22).Metyrosine dosed at 20 mg/kg was ineffective. Serial plasma sampling ofLPS-treated mice showed correlating inductions of catecholamines andcytokines in a time course of 3, 8 and 24 hours (FIG. 22). To documentthe generality of the effects of metyrosine, mice were treated with itprior to the induction of CRS by infection with parental C. novyi-NT. Itwas found that 85% of the mice pre-treated with metyrosine survivedwhile only 8% of the mice in the control arm survived (FIG. 23). Aspredicted, catecholamines and cytokines were substantially reduced inanimals pre-treated with metyrosine (FIG. 23).

To determine whether metyrosine could protect mice from bacterialinfections in general, its effects were evaluated when administeredprior to cecal ligation and puncture (CLP), a particularly challengingsepsis model. This puncture releases large numbers of enteric bacteria,including many species of gram-negative bacteria, into the peritoneum,causing polymicrobial peritoneal sepsis. Metyrosine significantlyreduced the mortality from the polymicrobial peritoneal sepsis—22% ofthe mice survived the acute phase, while all animals died in the controlarm (FIG. 23). However, when mice were pre-treated with metyrosine aswell as with the β-lactam antibiotic imipenem at 20 hours after CLP, >⅔of the mice survived CLP, while >90% of mice treated with the antibioticalone died. This experiment highlights the fact that death fromoverwhelming bacterial infections is due to two factors: the bacteriathemselves and the host reaction to the infection (i.e. CRS). Todemonstrate that the detrimental host response was diminished bymetyrosine pre-treatment, we measured the levels of circulatingcytokines as described above. Multiple cytokines characteristic ofsepsis or inflammation were substantially reduced by metyrosine afterinfection with C. novyi-NT or induction of polymicrobial peritonitis byCLP (FIG. 23). The expected effects of metyrosine on circulatingcatecholamines were also documented (FIG. 23).

To investigate which adrenergic receptor mediates the protective effect,adrenergic inhibitors prazosin, RX 821002, metoprolol, and ICI 118551were used to block the respective α1, α2, β1 and β2-adrenoceptors inmice treated with LPS. Blockade of α1 adrenergic receptors substantiallyreduced mortality and suppressed the production of catecholamines andcytokines, achieving results similar to those of metyrosine, whileblockade of α2, β1 and β2-adrenergic receptors did not reduce mortality(FIG. 24).

CRS is also observed after the administration of therapeutics notinvolving bacteria, and in particular with therapies that engage Tcells, including anti-T cell antibodies. Injection of an anti-murine CD3monoclonal antibody 145-2C11 into adult 5-6 months old BALB/c mice ledto a massive transient T-cell activation with high levels of IL-6,TNF-α, KC and MIP-2 and even death, recapitulating the CRS observed inhuman patients (FIG. 25). To determine whether catecholamines may alsodrive non-bacterial, T-cell antibody-related CRS, catecholamine levelswere first measured in mice 24 hours after injection of anti-CD3antibody. Indeed, the levels of epinephrine, norepinephrine and dopamineall increased substantially, along with cytokine release (FIG. 25).However, when mice were treated with metyrosine prior to administeringanti-CD3 antibody, the levels of circulating catecholamines weresignificantly reduced compared to the untreated controls. This decreasewas associated with major reductions of the pro-inflammatory cytokinesIL-6, TNF-α, KC, MIP-2 and MIP-1α, while IL-2 and IFN-γ were notsignificantly affected (FIG. 25). Most importantly, pre-treatment withmetyrosine significantly improved the survival of mice: the majority ofthose treated only with anti-CD3 antibodies died, while twelve of thefifteen mice pre-treated with metyrosine survived (FIG. 25). Theseresults were genetically substantiated in mice with myeloid-specificdeletion of TH, resulting in an impaired ability to synthesizecatecholamines. These mice (without metyrosine treatment) did notexhibit excessive cytokine release upon anti-CD3 exposure, mimickingwild-type mice pre-treated with metyrosine (FIG. 25).

To investigate whether CARTs can generate and release significantamounts of catecholamines during tumor cell killing, human Burkitt'slymphoma-derived CD19+ Raji cells were incubated with CD19-directedCARTs (hCART19) in vitro. For this purpose, primary donor T-cells weretransduced with mouse FMC63 anti-CD19 scFv containing a CD28-based hingeregion, transmembrane domain and costimulatory intracellular domainsfrom CD28 and 4-1BB coupled with the CD3ζ activation domain(CD19scFv-CD28-4-1BB-CD3ζ), as detailed in Methods. In vitro, cytolysisof Raji cells by hCART19 resulted in substantially increased levels ofepinephrine and norepinephrine as well as various cytokines such asIL-2, TNF-α, IFN-γ and MIP-1α in culture supernatants (FIGS. 27A-27B).To demonstrate the epinephrine-driven autocrine induction, co-culturedRaji and hCART19 cells were treated with epinephrine, which resulted inan amplified catecholamine and cytokine response (FIGS. 26A-26C).

To determine whether the autocrine epinephrine-induced production ofcatecholamines and cytokines takes place via new production rather thanthrough the release of preformed catecholamines and cytokines, proteinsynthesis inhibitor cycloheximide (CHX) was used to treat the hCART19cells during Raji exposure. It was found that the increase ofcatecholamines and cytokines (including TNF-α, MIP-1α, IFN-γ and GM-CSF)was greatly suppressed by treatment with CHX, suggesting that de novoprotein synthesis is required for their increased levels (FIGS.26D-26E).

Untransduced T-cells did not result in any significant changes to thecatecholamine or cytokine levels, unlike the situation with transduced(i.e., CART) cells (FIG. 27). Blockade of catecholamine synthesis withANP or metyrosine significantly decreased the hCART19-inducedcatecholamines and subsequent inflammatory responses as defined bycytokine production (FIG. 27).

To investigate the effect of catecholamine suppression onCART19-elicited CRS in vivo, an NSG™-SGM3 (NSGS) xenograft model wasemployed. In contrast to NSG mice that do not develop CRS, NSGS micethat express human myelo-supportive cytokines (IL3, GM-CSF and SCF)promote enhanced human T-cell engraftment and expansion and enablebetter modeling of T-cell associated diseases including CRS. NSGS micecan partially cause CRS (see, e.g., Wunderlich et al., 2010 Leukemia24:1785-1788; Sentman et al., 2016 J Immunol 197:4674-4685; Wunderlichet al., 2016 JCI Insight 1:e88181; and Gill et al., 2014 Blood123:2343-2354).

Cohorts of NSGS mice were first irradiated at a sublethal dose. The micewere IV injected with 10⁶ Raji cells one day later. Raji tumors wereallowed to grow for 6 days to the half time of the median survival ofuntreated mice to establish a condition in which CART cells would meet ahigh tumor burden and initiate lethal CRS within a few days, as iscommonly observed in patients (FIG. 27 and FIG. 28). Blood obtained at24 and 72 hours after hCART19 treatment revealed high levels ofcatecholamines (FIG. 27D and FIG. 28B), similar to what was observedafter administration of anti-CD3 antibodies to T-cells in theexperiments described above. This was accompanied by an acuteinflammatory response, as defined by significant elevations of varioushuman T-cell-produced cytokines in the circulation, including IL-2(hIL-2), IFN-γ (hIFN-γ), and TNF-α (hTNF-α) as well as the release ofendogenous host induced mouse cytokines IL-6, KC and MIP-2 consistentwith CRS seen in human patients (FIG. 27E and FIG. 28C). hCART19-treatedNSGS mice died 3-4 days after hCART19 injection with cytokine levels inthe blood that proved lethal in the anti-CD3 experiments (FIG. 27C).However, mice pre-treated with metyrosine showed significantly lowercatecholamine and cytokine levels (FIGS. 27D-27E and FIGS. 28B-28C),although mice eventually died with progressive lymphoma. Animals treatedwith the same amount of untransduced T-cells did not show any changes tosurvival, catecholamine or cytokine levels (FIG. 27 and FIG. 28).

The experiments described in FIGS. 27-28 were designed to assess theeffects of metyrosine on lethal toxicity associated with CRS followingCART therapy. It was conceivable that the reduction of catecholaminesand certain cytokines could interfere with the tumor response to CARTs,given the pleiotropic effects of catecholamines. To assess this issue, alow tumour burden model was employed to evaluate the impact ofcatecholamine blockage on the anti-tumor response (FIG. 29). hCART19could eradicate tumors under these conditions (FIG. 29) but could noteradicate the larger tumors described in FIG. 27. Bioluminescent imaging(BLI) was used to quantify the tumor burden over time after CARTinjection (FIG. 29). The lymphoma burden diminished within one weekafter hCART19 treatment in mice treated with metyrosine or controls, butsignificantly more so in the mice treated with metyrosine. Tumors wereeradicated, defined as BLI below 10⁶, in 6 out of 10 and 4 out of 10animals treated with the combination of hCART19 plus metyrosine andmeasured on day 6 and 19, respectively, compared to 0 out of 10 in theCART19 group, correlating with a long-term survival of 40% of themetyrosine-treated hCART19 mice versus 0% in hCART19 only group (FIG.29). Although none of the mice developed any clinical signs of toxicity,non-lethal elevation of catecholamines as well as human and mousecytokines were still present in the hCART19 cohort and reduced in themetyrosine-treated hCART19 cohort (FIG. 29). These experiments wererepeated with ANP, which also abrogated the hCART19-inducedcatecholamines and cytokine release in a similar way. Neither metyrosinenor ANP substantially interfered with in vivo CART cell expansion ortumor clearance (FIG. 28I and FIG. 29), and both were effective atpreventing CRS, as evidenced by the significant reduction in the levelsof mouse cytokines IL-6, KC and MIP-2 (FIG. 29).

Because available preclinical xenograft mouse models are poorlypredictive of the clinical behavior of CART cells, a second syngeneicmouse model was applied to assess whether antitumor activity might beaffected by CRS prophylaxis with metyrosine or ANP. For this aim,C57BL/6 mice were engrafted with Eμ-ALL cells that developedCD19-positive B-ALL. After verifying the establishment of the leukemictumor burden, mice were infused with mouse CART19 (mCART19) cellsdirected against mouse CD19 and containing the costimulatoryintracellular domain from CD28 coupled with the CD3ζ activation domain(m1928z), as detailed in Methods. Mice undergoing pharmacologicprophylaxis with ANP or metyrosine matched the therapeutic efficacy ofcontrol mice treated with mCART19 alone while the catecholamine andcytokine release was reduced (FIG. 30) thereby confirming that eitherdrug may prevent CRS while maintaining intact antitumor efficacy. Amodel explaining the reduced toxicity resulting from pre-treatment withmetyrosine is depicted in FIG. 15. Briefly, these data suggest thatcatecholamines triggers CRS via a self-amplifying feed-forward loop inimmune cells such as macrophages whereas myeloid-specific deletion of THwas protective.

This study provides evidence that catecholamines are drivers for CRS andthat enhanced production of catecholamines increases the intensity ofthe inflammatory response in bacterial and non-bacterial causes.Blockage of catecholamine synthesis reduced lethal cytokine levels intoa non-lethal range that not only ensured animal survival but also wasstill sufficient enough to allow effective tumor eradication.

OTHER EMBODIMENTS

It is to be understood that while the invention has been described inconjunction with the detailed description thereof, the foregoingdescription is intended to illustrate and not limit the scope of theinvention, which is defined by the scope of the appended claims. Otheraspects, advantages, and modifications are within the scope of thefollowing claims.

1. A method for preventing cytokine release in a mammal, wherein saidmethod comprises administering a catecholamine inhibitor to a mammalidentified as being at risk of developing cytokine release syndrome(CRS).
 2. The method of claim 1, wherein said CRS is associated withsepsis.
 3. The method of claim 1, wherein said CRS is associated with animmunotherapy.
 4. The method of claim 3, wherein said immunotherapy isselected from the group consisting of orthoclone OKT3, muromonab-CD3,rituximab, alemtuzumab, tosituzumab, CP-870,893, LO-CD2a/BTI-322,TGN1412, tisagenlecleucel, axicabtagene ciloleucel, bi-specific T-cellengagers (BiTEs), adoptive T-cell therapy, dendritic cell therapy,interferon therapy, interleukin therapy, bacterial therapy, and viraltherapy.
 5. The method of claim 3, wherein said immunotherapy is acancer immunotherapy.
 6. The method of claim 3, wherein saidimmunotherapy is for treating an autoimmune disease.
 7. The method ofclaim 6, wherein said autoimmune disease is selected from the groupconsisting of rheumatoid arthritis, juvenile idiopathic arthritis,ankylosing spondylitis, psoriasis, systemic lupus erythematosus, celiacdisease, type 1 diabetes, autoimmune encephalomyelitis, multiplesclerosis, central nervous system autoimmune demyelinating diseases,chronic inflammatory demyelinating polyneuropathy, transverse myelitis,polymyositis, dermatomyositis, Crohn's disease, ulcerative colitis,autoimmune hemolytic anemia, autoimmune cardiomyopathy, autoimmunethyroiditis, Graves' disease, Sjogren's syndrome, Goodpasture syndrome,autoimmune pancreatitis, Addison's disease, alopecia, myasthenia gravis,sarcoidosis, scleroderma, pemphigus vulgaris, mixed connective tissuedisease, bullous pemphigoid, and vitiligo.
 8. A method for inhibitingcatecholamine synthesis and/or catecholamine secretion in a mammal,wherein said method comprises administering a catecholamine inhibitor tosaid mammal.
 9. The method of claim 8, wherein said catecholamine isselected from the group consisting of epinephrine, norepinephrine,dopamine, and combinations thereof.
 10. The method of claim 9, whereinsaid catecholamine is epinephrine.
 11. A method for preventingtransplant rejection in a mammal, wherein said method comprisesadministering a catecholamine inhibitor to said mammal.
 12. The methodof claim 11, wherein said transplant rejection comprisesgraft-versus-host disease.
 13. The method of claim 1, wherein saidmammal is a human.
 14. The method of claim 1, wherein said catecholamineinhibitor comprises a tyrosine hydroxylase inhibitor, and wherein saidtyrosine hydroxylase inhibitor is metyrosine.
 15. The method of claim 1,wherein said catecholamine inhibitor comprises a natriuretic peptideselected from the group consisting of atrial natriuretic peptide (ANP),brain natriuretic peptide (BNP), C-type natriuretic peptide (CNP), anddendroaspis natriuretic peptide (DNP).
 16. The method of claim 15,wherein said natriuretic peptide is ANP, and wherein said ANP comprisesSEQ ID NO:1.
 17. The method claim 1, wherein said catecholamineinhibitor comprises an agent that can accelerate catecholaminedegradation, and wherein said agent that can accelerate catecholaminedegradation is a monoamine oxidase A activator or acatechol-O-methyltransferase (COMT) activator.
 18. The method of claim1, wherein said catecholamine inhibitor comprises an agent that canblock catecholamine release, wherein said agent that can blockcatecholamine release is gabapentin.
 19. The method of claim 1, whereinsaid catecholamine inhibitor comprises an agent that can block the α1adrenergic receptor, wherein said agent that can block the α1 adrenergicreceptor is prazosin.
 20. The method of claim 1, wherein saidcatecholamine inhibitor comprises both a natriuretic peptide and ahydroxylase inhibitor, wherein said natriuretic peptide is atrialnatriuretic peptide (ANP) and wherein said tyrosine hydroxylaseinhibitor is metyrosine.